Ciliates are eukaryotic unicellular organisms with complex morphology and developmental processes,including asexual and sexual processes.Conjugation is a form of sexual process that renews genetic materials.However,vi...Ciliates are eukaryotic unicellular organisms with complex morphology and developmental processes,including asexual and sexual processes.Conjugation is a form of sexual process that renews genetic materials.However,visualizing conjugation in ciliates is a challenge due to the complexity and dynamics of the process,while traditional staining methods are often insufficient for the research.This study introduces a new method for visualizing developmental progression in the nuclei during conjugation using Hoechst33342 staining.It describes how to proceed from cell culture,conjugation induction and synchronization,staining preparation,and observation to statistical analysis.The combination of fluorescent staining with the‘volume-fixing'technique eliminates the fixation and dehydration steps,thus reducing the overall operation time to just 20 minutes.This method offers several advantages over traditional staining techniques for studying the nuclei during conjugation.It improves image quality and workflow efficiency and enables real-time observation of live cell states.Potential solutions to challenges that may arise during experimental procedures are introduced and references and guidelines for cytological research are provided in this paper.展开更多
In the early days of deciphering the injured neuronal tissues led to the realization that contrast is necessary to discern the parts of the recovering tissues from the damaged ones.Early attempts relied on available(a...In the early days of deciphering the injured neuronal tissues led to the realization that contrast is necessary to discern the parts of the recovering tissues from the damaged ones.Early attempts relied on available(and often naturally occurring)staining substances.Incidentally,the active ingredients of most of them were small molecules.With the advent of time,the knowledge of chemistry helped identify compounds and conditions for staining.The staining reagents were even found to enhance the visibility of the organelles.Silver impregnation identification of Golgi bodies was discovered in owl optic nerve.Staining reagents since the late 1800s were widely used across all disciplines and for nerve tissue and became a key contributor to advancement in nerve-related research.The use of these reagents provided insight into the organization of the neuronal tissues and helped distinguish nerve degeneration from regeneration.The neuronal staining reagents have played a fundamental role in the clinical research facilitating the identification of biological mechanisms underlying eye and neuropsychiatric diseases.We found a lack of systematic description of all staining reagents,whether they had been used historically or currently used.There is a lack of readily available information for optimal staining of different neuronal tissues for a given purpose.We present here a grouping of the reagents based on their target location:(I)the central nervous system(CNS),(II)the peripheral nervous system(PNS),or(III)both.The biochemical reactions of most of the staining reagents is based on acidic or basic pH and specific reaction partners such as organelle or biomolecules that exists within the given tissue type.We present here a summary of the chemical composition,optimal staining condition,use for given neuronal tissue and,where possible,historic usage.Several biomolecules such as lipids and metabolites lack specific antibodies.Despite being non-specific the reagents enhance contrast and provide corroboration about the microenvironment.In future,these reagents in combination with emerging techniques such as imaging mass spectrometry and kinetic histochemistry will validate or expand our understanding of localization of molecules within tissues or cells that are important for ophthalmology and vision science.展开更多
In order to satisfy the need of rapid clinical diagnosis, a series of experimental studies for AgNOR (Argyrophilic NORassociated proteins) and DNA (Dereyribonucleic acid) staining was compared, from which a stable sta...In order to satisfy the need of rapid clinical diagnosis, a series of experimental studies for AgNOR (Argyrophilic NORassociated proteins) and DNA (Dereyribonucleic acid) staining was compared, from which a stable staining solution was selected to show mucin residence. Using the referred tables, with reference to the corresponding relation of the staining solution with temperature and time, reliable staining results for AgNOR and DNA could be obtained.These two modified staining methods are simple, reliable and easy to operate, able to provide good contrast for pathological diagnosis.展开更多
Objectives To develop a simple, accurate and reproducible method, which combines macro and histopathological techniques for determining the degree of lipid deposition in genetically modified mice. Method The entire a...Objectives To develop a simple, accurate and reproducible method, which combines macro and histopathological techniques for determining the degree of lipid deposition in genetically modified mice. Method The entire aortas from C57BL/6, ldlr-/- and apoE-/- mice were stained with Sudan IV using either in vivo perfusion or traditional in vitro enface staining techniques. Histological sections of aortic root and hearts were embedded in tissue freezing medium and cut with a cryostat, then stained with Oil Red O. The calculated aortic root area based on the aortic root circumference was used to reduce measurement errors. Results The in vitro en face staining can stain all fat, which include the adventitial tissue around aorta. However the in vivo perfusion staining can specifically stain the fatty deposition inside of aorta. Both entire aorta and aortic root section staining showed that there was a highly significant increase in fatty deposition in the aortas of the genetic modified mice. Although all mice genetic background was same, the apoE-/- mice had larger atherosclerotic lesions than ldlr-/- mice. Conclusions The new in vivo peffusion method is more accurate than the in vitro en face method. The combination of these macro and microscopic techniques overcomes the shortcomings of the earlier published methods which are generally limited to the measurement of fatty red staining areas only, neglecting non-specific adventitial fat staining around aorta and aortic root section tissue distortion.展开更多
Background: Tuberculosis is a highly infectious disease and India has the highest burden with it. Diagnosis of tuberculosis in many countries is still dependent on microscopy. Although its sensitivity is low in compar...Background: Tuberculosis is a highly infectious disease and India has the highest burden with it. Diagnosis of tuberculosis in many countries is still dependent on microscopy. Although its sensitivity is low in comparison to culture and molecular methods, its sensitivity can still be improved by using fluorescence staining method and processing of samples by homogenization and concentration method. Material and methods: Samples were collected from all newly registered suspected cases of tuberculosis in tertiary care hospital from outward and indoor department during a period of one year. Smears were prepared for Ziehl Neelsen stain and fluorescence stain both before and after homogenization and concentration procedure by 4% NAOH-2.9% sodium citrate method and results of them were interpreted according to RNTCP criteria for grading of sputum samples. All the samples were cultured in liquid culture MGIT system (Mycobacterial Growth Indicator Tube) and results of microscopy were compared with liquid culture taken as gold standard. Data were analyzed by using SPSS software version 16. Result: 350 samples were collected during study period. Out of 350 samples, 48 samples were positive for M. tuberculosis by MGIT system. In comparison with MGIT system, sensitivity of Z N stain for detection of acid fast bacilli was 77% before decontamination procedure, which was increased up to 85.42% after decontamination and concentration process. Sensitivity of fluroscence stain was 85.42% before processing, which was increased up to 91.67% after processing of samples. Conclusion: Sensitivity of smear microscopy can be enhanced by use of fluroscence microscopy and concentration method.展开更多
Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider uti...Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.展开更多
AIM： To detect Salmonella enteritidis （S. enteritidis） in paraffin slices and antigen location in infected duck tissues. METHODS： The rabbits were immunized with purified bacillus to obtain S. enteritidis-specific...AIM： To detect Salmonella enteritidis （S. enteritidis） in paraffin slices and antigen location in infected duck tissues. METHODS： The rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method （IFA） was established to detect the S. enteritidis antigen in paraffin slices. Detected S. enteritidis in each organ tissue of ducklings experimentally infected with S. enteritidis. RESULTS： The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen mainly distributed in the infected cell cytoplasm.CONCLUSION： IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.展开更多
AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in O...AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin (GSL); (3) Immunofluorescence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified-CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software. ' RESULTS: GSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovascularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group. CONCLUSION: GSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.展开更多
We developed a special methylene blue solution for staining of cervix shedding cells based on catalytic oxidizing chromogenic reaction, which shows a potential for cervical cancer cytology screening. We screened a tot...We developed a special methylene blue solution for staining of cervix shedding cells based on catalytic oxidizing chromogenic reaction, which shows a potential for cervical cancer cytology screening. We screened a total of 1 922 women for cervical cancer with the special methylene blue staining method and a conventional Pap smear method using cervix shedding cells. Then, the patients with positive indicators of the Pap smear or this special solution staining method were examined by the electron colposcopy and histopathological examination. Staining of cervical exfoliated cells with this reactive oxygen-based special solution showed that the number of positive cases was 140(7.28%). Among them, 21 cases showed dark green(1.09%), and 119 cases showed purple black(6.19%). The results of the Pap smear method showed that the number of positive cases was 123(6.40%), of which ASCUS was 105(5.46%), ASC-H was 5(0.26%), and LSIL was 9(0.47%), and HSIL was 4(0.21%). For cervical exfoliated cell special staining solution for screening cervical intraepithelial neoplasia(CIN-Ⅱ, CIN-Ⅲ) and cervical cancer, sensitivity was 83.33%, specificity was 65.52%, accuracy was 74.29%, missed diagnosis rate was 13.33%, positive coincidence rate was 51.43%, and the negative coincidence rate is 86.67%. Our results proved the value of this method for early screening of cervical cancer through clinical practice in China.展开更多
The solution chemical and optical characteristics of formation of amine-terminated polyamidoamine dendrimer G2.0（NH2-PAMAM G2.0）-Au nanocomposites in the aqueous solution of NH2-PAMAM G2.0 at various mole ratios of...The solution chemical and optical characteristics of formation of amine-terminated polyamidoamine dendrimer G2.0（NH2-PAMAM G2.0）-Au nanocomposites in the aqueous solution of NH2-PAMAM G2.0 at various mole ratios of Au（Ⅲ） to NH2-PAMAM G2.0 were studied by both UV-visible spectrometry and fluorospectrometry. The NH2-PAMAM G2.0-Au nanocomposites, with a type of structure in which one Au nanoparticle is surrounded by several NH2-PAMAM G2.0 dendrimers, emit strong bluish violet fluorescence, and are uniform, water soluble and biocompatible as well as very stable in frozen conditions. The size of gold nanoparticles in the nanocomposites is about 2.5 nm and decreases with the increase of NH2-PAMAM G2.0 concentration. The NH2-PAMAM G2.0 plays an important role in acting as host or micro-reactor for Au（Ⅲ） before Au（Ⅲ） reduction and acting as dispersant and stabilizer for gold nanoparticles after Au（Ⅲ） reduction. Preliminary experiments of cells staining to human embryonic lung fibroblast cell lines show that the NH2-PAMAM G2.0-Au nanocomposites can be used as optical imaging markers for bioanalyses and medical diagnoses.展开更多
In this study, mitotic metaphase chromosomes in mouse were identified by a new chromosome fluorescence banding technique combining DAPI staining with image analysis. Clear 4＇, 6-diamidino-2-phenylindole （DAPI） mult...In this study, mitotic metaphase chromosomes in mouse were identified by a new chromosome fluorescence banding technique combining DAPI staining with image analysis. Clear 4＇, 6-diamidino-2-phenylindole （DAPI） multiple bands like （J-hands could be produced in mouse. The Meta- Morph software was then used to generate linescans of pixel intensity for the banded chromosomes from short arm to long arm. These linescans were sufficient not only to identify each individual chromosome but also analyze the physical sites of bands in chromosome. Based on the results, the clear and accurate karyotype of mouse metaphase chromosomes was established. The technique is therefore considered to he a new method for cytological studies of mouse.展开更多
DNA-binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the va...DNA-binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei.These were:the cytoplasmic streaming in pollen tubes whose nuclei were stained,the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells,and the capability of isolated,prestained generative or sperm cells to fuse with other protoplasts.The results confirmed that 4',6-diamidino-2-phenylindole(DAPI),Hoechst 33258 and mithramycin could be used as real vital stains,though their efficiency varied from case to case;among them DAPI showed best effect.The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.展开更多
Seismic migration moves reflections to their true subsurface positions and yields seismic images of subsurface areas. However, due to limited acquisition aperture, complex overburden structure and target dipping angle...Seismic migration moves reflections to their true subsurface positions and yields seismic images of subsurface areas. However, due to limited acquisition aperture, complex overburden structure and target dipping angle, the migration often generates a distorted image of the actual subsurface structure. Seismic illumination and resolution analyses provide a quantitative description of how the above-mentioned factors distort the image. The point spread function （PSF） gives the resolution of the depth image and carries full information about the factors affecting the quality of the image. The staining algorithm establishes a correspondence between a certain structure and its relevant wavefield and reflected data. In this paper, we use the staining algorithm to calculate the PSFs, then use these PSFs for extracting the acquisition dip response and correcting the original depth image by deconvolution. We present relevant results of the SEG salt model. The staining algorithm provides an efficient tool for calculating the PSF and for conducting broadband seismic illumination and resolution analyses.展开更多
AIM: To evaluate clinicopathologic parameters and the clinical significance related lymphovascular invasion (LVI) by immunohistochemical staining (IHCS) in endoscopic submucosal dissection (ESD). METHODS: Between May ...AIM: To evaluate clinicopathologic parameters and the clinical significance related lymphovascular invasion (LVI) by immunohistochemical staining (IHCS) in endoscopic submucosal dissection (ESD). METHODS: Between May 2005 and May 2010, a total of 348 lesions from 321 patients (mean age 63 ± 10 years, men 74.6%) with early gastric cancer (EGC) who met indication criteria after ESD were analyzed retrospectively. The 348 lesions were divided into the absolute (n = 100, differentiated mucosal cancer without ulcer ≤ 20 mm) and expanded (n = 248) indica-tion groups after ESD. The 248 lesions were divided into four subgroups according to the expanded ESD indication. The presence of LVI was determined by factor Ⅷ-related antigen and D2-40 assessment. We compared LVI IHCS-negative group with LVI IHCSpositive in each group. RESULTS: LVI by hematoxylin-eosin staining (HES) and IHCS were all negative in the absolute group, while was observed in only the expanded groups. The positive rate of LVI by IHCS was higher than that of LVI by HES (n = 1, 0.4% vs n = 11, 4.4%, P = 0.044). LVI IHCS-positivity was observed when the cancer invaded to the mucosa 3 (M3) or submucosa 1 (SM1) levels, with a predominance of 63.6% in the subgroup that included only SM1 cancer (P < 0.01). In a univariate analysis, M3 or SM1 invasion by the tumor was significantly associated with a higher rate of LVI by IHCS, but no factor was significant in a multivariate analysis. There were no cases of tumor recurrence or metastasis during the median 26 mo follow-up. CONCLUSION: EGCs of the absolute group are immunohistochemically stable. The presence of LVI may be carefully examined by IHCS in an ESD expanded indication group with an invasion depth of M3 or greater.展开更多
The efficacies of some indigenous herbal dyes for use in staining plant materials were examined to obtain non-toxic, eco-friendly and cheap stains for use in plant histology. Dye extracts from Bixa orellana, Curcuma d...The efficacies of some indigenous herbal dyes for use in staining plant materials were examined to obtain non-toxic, eco-friendly and cheap stains for use in plant histology. Dye extracts from Bixa orellana, Curcuma domestica, Lonchocarpus cyanescens and Pterocarpus osun were used to stain wood sections using the existing standard staining procedures with little modification. All the extracts had affinity for the fibre and vessel elements except the extract from L. cyanescens. The extracts from C. domestica and B. orellana had higher selectivity than those ofP. osun for fibre. From the results of the absorbance curves, each of the dye extracts from all speciese had minimum of two peaks, indicating that they had two or more colour imparting chromophores except dye extract from C. domestica. All the dye extracts were acidic with pH range of 3.77 to 6.77. Therefore, this study shows that dye extracts from B. orellana, C. domestica and P. osun could be solitarily or in combination with artificial dyes for plant histological staining.展开更多
Objective To evaluate senile plaque formation and compare the sensitivity of three differentβ-amyloid(Aβ)labeling methods(antibody staining,Gallyas silver staining,and thioflavin-S staining)to detect Aβdeposition.M...Objective To evaluate senile plaque formation and compare the sensitivity of three differentβ-amyloid(Aβ)labeling methods(antibody staining,Gallyas silver staining,and thioflavin-S staining)to detect Aβdeposition.Methods APPswe/PSEN1dE9 transgenic mice(APP/PS1)of different ages were used to examine spatiotemporal changes in Aβplaque deposition.Antibody staining,Gallyas silver staining,and thioflavin-S staining were used to detect Aβplaque deposition in the same brain region of adjacent slices from model mice,and the results were compared.Results With aging,Aβplaques first appeared in the cortex and then the deposition increased throughout the whole brain.Significantly greater plaque deposition was detected by 6E10 antibody than that analyzed with Gallyas silver staining or thioflavin-S staining(P<0.05).Plaque deposition did not show significant difference between the APP/PS1 mice brains assayed with Gallyas silver staining and ones with thioflavin-S staining(P=0.0033).Conclusions The APP/PS1 mouse model of Alzheimer’s disease could mimick the progress of Aβplaques occurred in patients with Alzheimer’s disease.Antibody detection of Aβdeposition may be more sensitive than chemical staining methods.展开更多
Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical ...Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium（Rh123/PI）dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia.Methods：Twenty-five fertile men（with normal sperm parameters）and 230 infertile patients were examined.Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups：asthenospermia（n=30）and oligoasthenozoospermia（n=25）.Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function.Results：Significant differences were found between the normal and abnormal semen samples（P0.05）when Rh123＋/PI-,Rh123-/PI＋and Rh123-/PI-sperm were examined by FCM,but there was no significant difference between the asthenospermia（P=0.469） and oligoasthenozoospermia group（P=0.950）when Rh123＋/PI-and Rh123-/PI＋sperm were then examined;however,a significant difference was found between the 2 groups（P=0.003）when Rh123-/PI-sperm were examined.There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group,but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients（r=-0.509,-0.660;P=0.018,0.038）.Conclusion：Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.展开更多
AIM:To explore the value of Prussian blue staining in the diagnosis of ocular siderosis.METHODS:Between January 2012 and January 2013,the Prussian blue stain used in anterior lens capsule and vitreous liquid after cen...AIM:To explore the value of Prussian blue staining in the diagnosis of ocular siderosis.METHODS:Between January 2012 and January 2013,the Prussian blue stain used in anterior lens capsule and vitreous liquid after centrifugation from patients with definitive diagnosis and suspicious diagnosed of ocular siderosis. At the same time, give a negative control.RESULTS:Anterior lens capsule membrane and liquid of vitreous cavity from patients with definitive diagnosis and suspicious diagnosed of ocular siderosis revealed ferric ions that stained positively with Prussian blue. In the control group, there is no positive reaction.CONCLUSION:Prussian blue staining in the diagnosis of ocular siderosis has a very significant worth,suspected cases can be definitive diagnosed.展开更多
Negative staining is an effective method that can be used for electron microscopic study to observe fine structural morphology without destruction of bacterial structure. Although uranium acetate is used worldwide as ...Negative staining is an effective method that can be used for electron microscopic study to observe fine structural morphology without destruction of bacterial structure. Although uranium acetate is used worldwide as a general dyeing solution, it is extremely difficult to use it by a new purchase at a research institution because it falls under the nuclear regulation substance in Japan. Therefore, we examined alternative reagents for negative staining that could replace uranium acetate through bacterial observation with an electron microscope. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes were examined by four stain reagents (phosphotungstic acid (PTA), EMstainer, TI blue, and uranium acetate). Pre cultured bacteria were stained with each stain reagents on a copper grid, washed with PBS, and observed with a transmission electron microscope. In the comparison between bacterial structures, the cell wall structure and bacterial flagella could be observed well in the order of PTA, EMstainer, and uranium acetate. With TI blue staining, flagella could be observed very poorly. In comparison between bacteria, gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, could be observed well as compared with gram positive cocci such as Staphylococcus aureus and Streptococcus pyogenes. The uranium acetate looked very coarse in background particles. Since crystals tend to precipitate, TI blue also required filtering, and electron beams were absorbed by the agglomerated crystals, and the frequency of electronic burning occurred high frequency. In this study, there was clear difference in the observation conditions depending on the type of bacteria and the kind of the staining reagents. Especially, it was confirmed that good negative staining features of Pseudomonas aeruginosa by electron microscope were obtained by PTA and EMstainer staining. These alternative reagents are considered to be a candidate for a negative staining.展开更多
A detailed morphological study of neurons in healthy and pathological conditions requires reasonably a number of special techniques, which may visualize the majority of neu- rons in a thick three-dimensional arrangeme...A detailed morphological study of neurons in healthy and pathological conditions requires reasonably a number of special techniques, which may visualize the majority of neu- rons in a thick three-dimensional arrangement. A detailed visualization of neurons must include the cell body, most of the dendritic arbor, the dendritic spines, the axon, the axonal collaterals and the synapses. An ideal morphological technique for the study of degeneration and regeneration processes of the central nervous system must also visualize clearly the long and short neuronal circuits, as well as the dendritic and axonal bands and tracks.展开更多
基金supported by the National Natural Science Foundation of China(No.32270558)。
文摘Ciliates are eukaryotic unicellular organisms with complex morphology and developmental processes,including asexual and sexual processes.Conjugation is a form of sexual process that renews genetic materials.However,visualizing conjugation in ciliates is a challenge due to the complexity and dynamics of the process,while traditional staining methods are often insufficient for the research.This study introduces a new method for visualizing developmental progression in the nuclei during conjugation using Hoechst33342 staining.It describes how to proceed from cell culture,conjugation induction and synchronization,staining preparation,and observation to statistical analysis.The combination of fluorescent staining with the‘volume-fixing'technique eliminates the fixation and dehydration steps,thus reducing the overall operation time to just 20 minutes.This method offers several advantages over traditional staining techniques for studying the nuclei during conjugation.It improves image quality and workflow efficiency and enables real-time observation of live cell states.Potential solutions to challenges that may arise during experimental procedures are introduced and references and guidelines for cytological research are provided in this paper.
基金supported by an unrestricted grant from Research to Prevent Blindness and NIH grants EY14801,EY031292.
文摘In the early days of deciphering the injured neuronal tissues led to the realization that contrast is necessary to discern the parts of the recovering tissues from the damaged ones.Early attempts relied on available(and often naturally occurring)staining substances.Incidentally,the active ingredients of most of them were small molecules.With the advent of time,the knowledge of chemistry helped identify compounds and conditions for staining.The staining reagents were even found to enhance the visibility of the organelles.Silver impregnation identification of Golgi bodies was discovered in owl optic nerve.Staining reagents since the late 1800s were widely used across all disciplines and for nerve tissue and became a key contributor to advancement in nerve-related research.The use of these reagents provided insight into the organization of the neuronal tissues and helped distinguish nerve degeneration from regeneration.The neuronal staining reagents have played a fundamental role in the clinical research facilitating the identification of biological mechanisms underlying eye and neuropsychiatric diseases.We found a lack of systematic description of all staining reagents,whether they had been used historically or currently used.There is a lack of readily available information for optimal staining of different neuronal tissues for a given purpose.We present here a grouping of the reagents based on their target location:(I)the central nervous system(CNS),(II)the peripheral nervous system(PNS),or(III)both.The biochemical reactions of most of the staining reagents is based on acidic or basic pH and specific reaction partners such as organelle or biomolecules that exists within the given tissue type.We present here a summary of the chemical composition,optimal staining condition,use for given neuronal tissue and,where possible,historic usage.Several biomolecules such as lipids and metabolites lack specific antibodies.Despite being non-specific the reagents enhance contrast and provide corroboration about the microenvironment.In future,these reagents in combination with emerging techniques such as imaging mass spectrometry and kinetic histochemistry will validate or expand our understanding of localization of molecules within tissues or cells that are important for ophthalmology and vision science.
文摘In order to satisfy the need of rapid clinical diagnosis, a series of experimental studies for AgNOR (Argyrophilic NORassociated proteins) and DNA (Dereyribonucleic acid) staining was compared, from which a stable staining solution was selected to show mucin residence. Using the referred tables, with reference to the corresponding relation of the staining solution with temperature and time, reliable staining results for AgNOR and DNA could be obtained.These two modified staining methods are simple, reliable and easy to operate, able to provide good contrast for pathological diagnosis.
基金supported by the Hong Kong WangKuan Cheng Foundation GrantBritish Heart Foundation
文摘Objectives To develop a simple, accurate and reproducible method, which combines macro and histopathological techniques for determining the degree of lipid deposition in genetically modified mice. Method The entire aortas from C57BL/6, ldlr-/- and apoE-/- mice were stained with Sudan IV using either in vivo perfusion or traditional in vitro enface staining techniques. Histological sections of aortic root and hearts were embedded in tissue freezing medium and cut with a cryostat, then stained with Oil Red O. The calculated aortic root area based on the aortic root circumference was used to reduce measurement errors. Results The in vitro en face staining can stain all fat, which include the adventitial tissue around aorta. However the in vivo perfusion staining can specifically stain the fatty deposition inside of aorta. Both entire aorta and aortic root section staining showed that there was a highly significant increase in fatty deposition in the aortas of the genetic modified mice. Although all mice genetic background was same, the apoE-/- mice had larger atherosclerotic lesions than ldlr-/- mice. Conclusions The new in vivo peffusion method is more accurate than the in vitro en face method. The combination of these macro and microscopic techniques overcomes the shortcomings of the earlier published methods which are generally limited to the measurement of fatty red staining areas only, neglecting non-specific adventitial fat staining around aorta and aortic root section tissue distortion.
文摘Background: Tuberculosis is a highly infectious disease and India has the highest burden with it. Diagnosis of tuberculosis in many countries is still dependent on microscopy. Although its sensitivity is low in comparison to culture and molecular methods, its sensitivity can still be improved by using fluorescence staining method and processing of samples by homogenization and concentration method. Material and methods: Samples were collected from all newly registered suspected cases of tuberculosis in tertiary care hospital from outward and indoor department during a period of one year. Smears were prepared for Ziehl Neelsen stain and fluorescence stain both before and after homogenization and concentration procedure by 4% NAOH-2.9% sodium citrate method and results of them were interpreted according to RNTCP criteria for grading of sputum samples. All the samples were cultured in liquid culture MGIT system (Mycobacterial Growth Indicator Tube) and results of microscopy were compared with liquid culture taken as gold standard. Data were analyzed by using SPSS software version 16. Result: 350 samples were collected during study period. Out of 350 samples, 48 samples were positive for M. tuberculosis by MGIT system. In comparison with MGIT system, sensitivity of Z N stain for detection of acid fast bacilli was 77% before decontamination procedure, which was increased up to 85.42% after decontamination and concentration process. Sensitivity of fluroscence stain was 85.42% before processing, which was increased up to 91.67% after processing of samples. Conclusion: Sensitivity of smear microscopy can be enhanced by use of fluroscence microscopy and concentration method.
基金Acknowledgments This study was supported by grants from the China National Natural Science Foundation （Nos. 30430530 and 30571337） and from the Momentous Research Project of the China Ministry of Science and Technology （No. 2006CB944003）.
文摘Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.
基金the National Key Technology R&D Program of China, No. 2004BA 901A 03National Scientific and Sechnical Support Program, No. 2007Z06-017+3 种基金The Cultvation Fund of the Key Scientific and Technical Innovation Project & Ministry of Education of China, No. 706050Program for New Century Excellent Talents in University, No. NCET-04-0906/NCET-06-0818Sichuan Province Basic Research Program, No. 04JY0290061/07JY029-017Program for Key Disciplines Construction of Sichuan Province No. SZD0418
文摘AIM： To detect Salmonella enteritidis （S. enteritidis） in paraffin slices and antigen location in infected duck tissues. METHODS： The rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method （IFA） was established to detect the S. enteritidis antigen in paraffin slices. Detected S. enteritidis in each organ tissue of ducklings experimentally infected with S. enteritidis. RESULTS： The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen mainly distributed in the infected cell cytoplasm.CONCLUSION： IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.
基金Supported by National Natural Science Foundation of China(No.30973899)
文摘AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin (GSL); (3) Immunofluorescence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified-CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software. ' RESULTS: GSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovascularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group. CONCLUSION: GSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.
基金Supported by the Joint Foundation from Health Commission of Hubei Province(WJ2019H007)
文摘We developed a special methylene blue solution for staining of cervix shedding cells based on catalytic oxidizing chromogenic reaction, which shows a potential for cervical cancer cytology screening. We screened a total of 1 922 women for cervical cancer with the special methylene blue staining method and a conventional Pap smear method using cervix shedding cells. Then, the patients with positive indicators of the Pap smear or this special solution staining method were examined by the electron colposcopy and histopathological examination. Staining of cervical exfoliated cells with this reactive oxygen-based special solution showed that the number of positive cases was 140(7.28%). Among them, 21 cases showed dark green(1.09%), and 119 cases showed purple black(6.19%). The results of the Pap smear method showed that the number of positive cases was 123(6.40%), of which ASCUS was 105(5.46%), ASC-H was 5(0.26%), and LSIL was 9(0.47%), and HSIL was 4(0.21%). For cervical exfoliated cell special staining solution for screening cervical intraepithelial neoplasia(CIN-Ⅱ, CIN-Ⅲ) and cervical cancer, sensitivity was 83.33%, specificity was 65.52%, accuracy was 74.29%, missed diagnosis rate was 13.33%, positive coincidence rate was 51.43%, and the negative coincidence rate is 86.67%. Our results proved the value of this method for early screening of cervical cancer through clinical practice in China.
文摘The solution chemical and optical characteristics of formation of amine-terminated polyamidoamine dendrimer G2.0（NH2-PAMAM G2.0）-Au nanocomposites in the aqueous solution of NH2-PAMAM G2.0 at various mole ratios of Au（Ⅲ） to NH2-PAMAM G2.0 were studied by both UV-visible spectrometry and fluorospectrometry. The NH2-PAMAM G2.0-Au nanocomposites, with a type of structure in which one Au nanoparticle is surrounded by several NH2-PAMAM G2.0 dendrimers, emit strong bluish violet fluorescence, and are uniform, water soluble and biocompatible as well as very stable in frozen conditions. The size of gold nanoparticles in the nanocomposites is about 2.5 nm and decreases with the increase of NH2-PAMAM G2.0 concentration. The NH2-PAMAM G2.0 plays an important role in acting as host or micro-reactor for Au（Ⅲ） before Au（Ⅲ） reduction and acting as dispersant and stabilizer for gold nanoparticles after Au（Ⅲ） reduction. Preliminary experiments of cells staining to human embryonic lung fibroblast cell lines show that the NH2-PAMAM G2.0-Au nanocomposites can be used as optical imaging markers for bioanalyses and medical diagnoses.
文摘In this study, mitotic metaphase chromosomes in mouse were identified by a new chromosome fluorescence banding technique combining DAPI staining with image analysis. Clear 4＇, 6-diamidino-2-phenylindole （DAPI） multiple bands like （J-hands could be produced in mouse. The Meta- Morph software was then used to generate linescans of pixel intensity for the banded chromosomes from short arm to long arm. These linescans were sufficient not only to identify each individual chromosome but also analyze the physical sites of bands in chromosome. Based on the results, the clear and accurate karyotype of mouse metaphase chromosomes was established. The technique is therefore considered to he a new method for cytological studies of mouse.
文摘DNA-binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei.These were:the cytoplasmic streaming in pollen tubes whose nuclei were stained,the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells,and the capability of isolated,prestained generative or sperm cells to fuse with other protoplasts.The results confirmed that 4',6-diamidino-2-phenylindole(DAPI),Hoechst 33258 and mithramycin could be used as real vital stains,though their efficiency varied from case to case;among them DAPI showed best effect.The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.
基金funded by the National Natural Science Foundation of China(No.41374006 and 41274117)
文摘Seismic migration moves reflections to their true subsurface positions and yields seismic images of subsurface areas. However, due to limited acquisition aperture, complex overburden structure and target dipping angle, the migration often generates a distorted image of the actual subsurface structure. Seismic illumination and resolution analyses provide a quantitative description of how the above-mentioned factors distort the image. The point spread function （PSF） gives the resolution of the depth image and carries full information about the factors affecting the quality of the image. The staining algorithm establishes a correspondence between a certain structure and its relevant wavefield and reflected data. In this paper, we use the staining algorithm to calculate the PSFs, then use these PSFs for extracting the acquisition dip response and correcting the original depth image by deconvolution. We present relevant results of the SEG salt model. The staining algorithm provides an efficient tool for calculating the PSF and for conducting broadband seismic illumination and resolution analyses.
文摘AIM: To evaluate clinicopathologic parameters and the clinical significance related lymphovascular invasion (LVI) by immunohistochemical staining (IHCS) in endoscopic submucosal dissection (ESD). METHODS: Between May 2005 and May 2010, a total of 348 lesions from 321 patients (mean age 63 ± 10 years, men 74.6%) with early gastric cancer (EGC) who met indication criteria after ESD were analyzed retrospectively. The 348 lesions were divided into the absolute (n = 100, differentiated mucosal cancer without ulcer ≤ 20 mm) and expanded (n = 248) indica-tion groups after ESD. The 248 lesions were divided into four subgroups according to the expanded ESD indication. The presence of LVI was determined by factor Ⅷ-related antigen and D2-40 assessment. We compared LVI IHCS-negative group with LVI IHCSpositive in each group. RESULTS: LVI by hematoxylin-eosin staining (HES) and IHCS were all negative in the absolute group, while was observed in only the expanded groups. The positive rate of LVI by IHCS was higher than that of LVI by HES (n = 1, 0.4% vs n = 11, 4.4%, P = 0.044). LVI IHCS-positivity was observed when the cancer invaded to the mucosa 3 (M3) or submucosa 1 (SM1) levels, with a predominance of 63.6% in the subgroup that included only SM1 cancer (P < 0.01). In a univariate analysis, M3 or SM1 invasion by the tumor was significantly associated with a higher rate of LVI by IHCS, but no factor was significant in a multivariate analysis. There were no cases of tumor recurrence or metastasis during the median 26 mo follow-up. CONCLUSION: EGCs of the absolute group are immunohistochemically stable. The presence of LVI may be carefully examined by IHCS in an ESD expanded indication group with an invasion depth of M3 or greater.
文摘The efficacies of some indigenous herbal dyes for use in staining plant materials were examined to obtain non-toxic, eco-friendly and cheap stains for use in plant histology. Dye extracts from Bixa orellana, Curcuma domestica, Lonchocarpus cyanescens and Pterocarpus osun were used to stain wood sections using the existing standard staining procedures with little modification. All the extracts had affinity for the fibre and vessel elements except the extract from L. cyanescens. The extracts from C. domestica and B. orellana had higher selectivity than those ofP. osun for fibre. From the results of the absorbance curves, each of the dye extracts from all speciese had minimum of two peaks, indicating that they had two or more colour imparting chromophores except dye extract from C. domestica. All the dye extracts were acidic with pH range of 3.77 to 6.77. Therefore, this study shows that dye extracts from B. orellana, C. domestica and P. osun could be solitarily or in combination with artificial dyes for plant histological staining.
基金Supported by the 2016 Major Collaborative Innovation Program of the Chinese Academy of Medical Sciences(2016-I2M-1004)
文摘Objective To evaluate senile plaque formation and compare the sensitivity of three differentβ-amyloid(Aβ)labeling methods(antibody staining,Gallyas silver staining,and thioflavin-S staining)to detect Aβdeposition.Methods APPswe/PSEN1dE9 transgenic mice(APP/PS1)of different ages were used to examine spatiotemporal changes in Aβplaque deposition.Antibody staining,Gallyas silver staining,and thioflavin-S staining were used to detect Aβplaque deposition in the same brain region of adjacent slices from model mice,and the results were compared.Results With aging,Aβplaques first appeared in the cortex and then the deposition increased throughout the whole brain.Significantly greater plaque deposition was detected by 6E10 antibody than that analyzed with Gallyas silver staining or thioflavin-S staining(P<0.05).Plaque deposition did not show significant difference between the APP/PS1 mice brains assayed with Gallyas silver staining and ones with thioflavin-S staining(P=0.0033).Conclusions The APP/PS1 mouse model of Alzheimer’s disease could mimick the progress of Aβplaques occurred in patients with Alzheimer’s disease.Antibody detection of Aβdeposition may be more sensitive than chemical staining methods.
基金supported by the program of The Project Supported by Natural Science Basic Research Plan in Shaanxi Province of China(No.SJ08-ZD05)
文摘Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium（Rh123/PI）dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia.Methods：Twenty-five fertile men（with normal sperm parameters）and 230 infertile patients were examined.Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups：asthenospermia（n=30）and oligoasthenozoospermia（n=25）.Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function.Results：Significant differences were found between the normal and abnormal semen samples（P0.05）when Rh123＋/PI-,Rh123-/PI＋and Rh123-/PI-sperm were examined by FCM,but there was no significant difference between the asthenospermia（P=0.469） and oligoasthenozoospermia group（P=0.950）when Rh123＋/PI-and Rh123-/PI＋sperm were then examined;however,a significant difference was found between the 2 groups（P=0.003）when Rh123-/PI-sperm were examined.There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group,but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients（r=-0.509,-0.660;P=0.018,0.038）.Conclusion：Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.
基金Supported by Education Department Funding of Sichuan Province,China(No.2005B020)
文摘AIM:To explore the value of Prussian blue staining in the diagnosis of ocular siderosis.METHODS:Between January 2012 and January 2013,the Prussian blue stain used in anterior lens capsule and vitreous liquid after centrifugation from patients with definitive diagnosis and suspicious diagnosed of ocular siderosis. At the same time, give a negative control.RESULTS:Anterior lens capsule membrane and liquid of vitreous cavity from patients with definitive diagnosis and suspicious diagnosed of ocular siderosis revealed ferric ions that stained positively with Prussian blue. In the control group, there is no positive reaction.CONCLUSION:Prussian blue staining in the diagnosis of ocular siderosis has a very significant worth,suspected cases can be definitive diagnosed.
文摘Negative staining is an effective method that can be used for electron microscopic study to observe fine structural morphology without destruction of bacterial structure. Although uranium acetate is used worldwide as a general dyeing solution, it is extremely difficult to use it by a new purchase at a research institution because it falls under the nuclear regulation substance in Japan. Therefore, we examined alternative reagents for negative staining that could replace uranium acetate through bacterial observation with an electron microscope. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes were examined by four stain reagents (phosphotungstic acid (PTA), EMstainer, TI blue, and uranium acetate). Pre cultured bacteria were stained with each stain reagents on a copper grid, washed with PBS, and observed with a transmission electron microscope. In the comparison between bacterial structures, the cell wall structure and bacterial flagella could be observed well in the order of PTA, EMstainer, and uranium acetate. With TI blue staining, flagella could be observed very poorly. In comparison between bacteria, gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, could be observed well as compared with gram positive cocci such as Staphylococcus aureus and Streptococcus pyogenes. The uranium acetate looked very coarse in background particles. Since crystals tend to precipitate, TI blue also required filtering, and electron beams were absorbed by the agglomerated crystals, and the frequency of electronic burning occurred high frequency. In this study, there was clear difference in the observation conditions depending on the type of bacteria and the kind of the staining reagents. Especially, it was confirmed that good negative staining features of Pseudomonas aeruginosa by electron microscope were obtained by PTA and EMstainer staining. These alternative reagents are considered to be a candidate for a negative staining.
文摘A detailed morphological study of neurons in healthy and pathological conditions requires reasonably a number of special techniques, which may visualize the majority of neu- rons in a thick three-dimensional arrangement. A detailed visualization of neurons must include the cell body, most of the dendritic arbor, the dendritic spines, the axon, the axonal collaterals and the synapses. An ideal morphological technique for the study of degeneration and regeneration processes of the central nervous system must also visualize clearly the long and short neuronal circuits, as well as the dendritic and axonal bands and tracks.
