Comparison of Hematoxylin-eosin Staining and Methyl Violet Staining for Displaying Ghost Cells

Purpose:.To compare the merits and limitations of hematoxylin-eosin(HE) and methyl violet staining for displaying ghost cells from vitreous or aqueous humor.Methods:.A specimen containing ghost cells was adjusted to five different concentrations:(12×104,.10×104,.8×104, 6×104and 4×104cells / ml) and subjected to smearing and methyl violet and HE staining..The staining results were observed by light microscopy.Results: The ghost cells were readily observed at a cell density of > 8×104cells / ml with methyl violet staining,.but only a few cells were occasionally seen at lower cell densities..In contrast,.ghost cells were seen at all cell densities with HE staining.Conclusion: Methyl violet staining is more rapid and simpler for the identification of ghost cells, but its staining color more readily fades, the slides cannot be stored, and it is only effective at a cell density of > 8 ×104cells / ml. In contrast,.HE staining is more time-consuming but it can display cell morphology and distinguish cell components more explicitly and slides can be permanently stored. HE staining has advantages over methyl violet staining in detecting the ghost cells when the concentration is < 8×104cells / ml.

A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy

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.

A revisit to staining reagents for neuronal tissues

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.

Mouse Karyotype Obtained by Combining DAPI Staining with Image Analysis

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.

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

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.

Application of an indirect immunofluorescent staining method for detection of Salmonella enteritidis in paraffin slices and antigen location in infected duck tissues

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.

Comparison ofβ-Amyloid Plaque Labeling Methods:Antibody Staining,Gallyas Silver Staining,and Thioflavin-S Staining

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.

Application of Prussian blue staining in the diagnosis of ocular siderosis

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.

Preparation, optical properties and cell staining of water soluble amine-terminated PAMAM G2.0-Au nanocomposites

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.

Screening of some indigenous herbal dyes for use in plant histological staining

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.

Evaluation of sperm mitochondrial function using rh123/PI dual fluorescent staining in asthenospermia and oligoasthenozoospermia

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.

Pollen viability of Polygala paniculata L.(Polygalaceae)using different staining methods

Polygala paniculata L.is a medicinal plant that grows in the Brazilian Atlantic coast,known as‘barba-de-São-João’,‘barba-de-bode’,‘vassourinha branca’,and‘mimosa’.In this study,pollen viability was estimated by three different staining methods:2%acetic orcein,2%acetic carmine,and Alexander’s stain.The young inflorescences of twenty accessions were collected and fixed in a solution of ethanol:acetic acid(3:1)for 24 hours,then stored in ethanol 70%under refrigeration.Six slides per plant,two for each stain,were prepared by squashing,and 300 pollen grains per slide were analyzed.Pollen viability was high(>70%)for most accessions of P.paniculata using the Alexander’s stain,which proved the most adequate method to estimate pollen viability.

Broadband seismic illumination and resolution analyses based on staining algorithm

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.

Fluorescent vital staining of plant sexual cell nuclei with DNA-specific fluorochromes and its application in gametoplast fusion

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.

Effect of Intravital Staining on Leaf Surface Coloring and Plant Carbon and Nitrogen Nutrition in Chlorophytum comosum var. variegatum

Using potted seedlings of Chlorophytum comosum var. variegatum as the experimental materials, the effect of 2.0 mmol/L methyl orange （ Treatment T1 ）, 1.0 mmol/L methyl violet （ Treatment T2 ） and 1.0 mmol/L neutral red （ Treatment T3 ） on the biomass, root-shoot ratio, leaf color indices, plant carbon and nitrogen nutrition were studied. The results showed that the biomass of Treatment T3 was significantly greater than that of treatments T1 and CK. The root-shoot ratio decreased significantly in treatments T1, T2 and T3 , and the decrease in T3 was most obvious. In all the three treatments with coloring agent, a ^＊ , b ^＊ and L ^＊ values were increased gradually, C value were decreased, H0 and CIRG were increased, and the leaves were pink. In addition, the contents of chlorophyll a, chlorophyll b, chlorophyll a ＋ b and carotenoid were significantly decreased. The contents of soluble sugar and starch were also decreased, and the decrease in Treatment T2 was most significant. The contents of soluble protein and total nitrogen were increased, and the increase was most dramatic in Treatment T3. The carbon to nitrogen ratio was decreased. The results proved that staining can improve the ornamental value of indoor plants, despite its effects on plant carbon and nitrogen nutrition of C. comosum vat. variegatum, dyeing.

Emerging Advances to Transform Histopathology Using Virtual Staining

In an age where digitization is widespread in clinical and preclinical workflows,pathology is still predominantly practiced by microscopic evaluation of stained tissue specimens affixed on glass slides.Over the last decade,new high throughput digital scanning microscopes have ushered in the era of digital pathology that,along with recent advances in machine vision,have opened up new possibilities for Computer-Aided-Diagnoses.Despite these advances,the high infrastructural costs related to digital pathology and the perception that the digitization process is an additional and nondirectly reimbursable step have challenged its widespread adoption.Here,we discuss how emerging virtual staining technologies and machine learning can help to disrupt the standard histopathology workflow and create new avenues for the diagnostic paradigm that will benefit patients and healthcare systems alike via digital pathology.

Optimization of DNA Staining Technology for Development of Autonomous Microbe Sensor for Injection Seawater Systems

Microbial activity in the water injection system in oil and gas industry leads to an array of challenges, including biofouling, injectivity loss, reservoir plugging, and microbiologically influenced corrosion (MIC). An effective mitigation strategy requires online and real-time monitoring of microbial activity and growth in the system so that the operators can apply and adjust counter-measures quickly and properly. The previous study [1] identified DNA staining technology-with PicoGreen and SYBR Green dyes—as a very promising method for automated, online determination of microbial cell abundance in the vast Saudi Aramco injection seawater systems. This study evaluated DNA staining technology on detection limit, automation potential, and temperature stability for the construction of automated sensor prototype. DNA staining with SYBR Green dye was determined to be better suited for online and real-time monitoring of microbial activity in the Saudi Aramco seawater systems. SYBR Green staining does not require sample pre-treatment, and the fluorescence signal intensity is more stable at elevated temperatures up to 30℃. The lower detection limit of 2 × 103/ml was achieved under the optimized conditions, which is sufficient to detect microbial numbers in Saudi Aramco injection seawater. Finally, the requirements for design and construction of SYBR-based automated sensor prototype were determined.

Does immunohistochemical staining have a clinical impact in early gastric cancer conducted endoscopic submucosal dissection?

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.

Staining neurons with Golgi techniques in degenerative diseases ofthe brain

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.

Simplified heavy metal staining techniques demonstrated with Fast Plant leaf tissue

Fast Plant(Brassica rapa,Cruciferae)leaf tissuefixed in glutaraldehyde-acrolein and post-fixed in os-mium,was examined for response to several easily-prepared heavy metal stains.Lead and uranium,separately and in combination,gave typical resultsacross the spectrum of cell organellets.As a single stainfollowing osmium,bismuth produced images seeminglyequivalent to lead and uranium.Phosphotungstic acidproduced very good membrane delineation but produceda washed-out background image similar to that from leadstaining.Carbohydrate compounds were especiallyresponsive to ruthenium;the cytoplasm and the matrixof all organelles were also stained very well.Theprocedures were no more demanding than traditionalstaining methods and may be easily used in research andteaching.Fast Plant materials are a reliable,quick andeasy source of living material.