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.

A RAPID STAINING METHOD SHOWING AgNOR AND DNA IN PATHOLOGICAL DIAGNOSIS

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.

Comparative Study of Z N Staining vs. Flurochrome Staining and Impact of Sample Processing on Diagnosis of Tuberculosis from Various Clinical Samples

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.

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.

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.

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.

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.

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.

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.

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.

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.

Application of Luxol Fast Blue staining in locating the corticospinal tract in adult rats

BACKGROUND: There are many methods for myelin staining, mordant, or oil-soluble dye or the special reaction of osmic acid with lipoid is used according to different principles. The commonly used methods are classic Well staining, classic lithium carbonate-haematine staining, fast green staining, silver staining, etc. Luxol Fast Blue can brightly stain myelin sheath, and has certain specificity. The background can be very clean if there is proper differentiation, whereas Luxol Fast Blue is cheap and convenient to operate, thus it is an ideal staining reagent for routine myelin sheath. OBJECTIVE: To show the corticospinal tract of normal adult rats with Luxol Fast Blue staining method. DESIGN: A repetitive measurement design. SETTINGS: Institute of Nuerobiology, Nantong University; Department of Rehabilitation Medicine, Affiliated Hospital of Nantong University. MATERIALS: Six healthy adult male SD rats of clean degree, weighing averagely 300 g, were provided by the experimental animal center of Nantong University. 1 g/L Luxol Fast Blue solution was provided by Sigma Company; Leica CM1900 cryostat microtome by Leica Company; Leica DMR microscope by Leica Company. METHODS: The experiment was carried out in the Staff Room of Human Anatomy, Nantong University in May 2005. The rats were given intraperitoneal injection of combined anesthetic (2 mL/kg), then the chest was open for perfusing saline and phosphate buffer containing formamint via heart. Brain and spinal cord were removed after 1 hour then fixed, then changed to phosphate buffer (pH 7.4) containing 300 g/L saccharu at 4 ℃, and stayed overnight, tissue blocks at pyramid, decussation of pyramid and cervical, thoracic, lumbar and sacral segments of spinal cord were removed to prepare continuous horizontal frozen sections (30 μm) after sedimentation, the sections were dried at room temperature. The corticospinal tract of normal adult rats were shown with Luxol Fast Blue staining method, and observed under Leica DMR microscope. MAIN OUTCOME MEASURES: Positive fibers in Luxol Fast Blue staining. RESULTS: After the Luxol Fast Blue staining, the labeled myelinated nerve fibers were bright blue. They located in the pyramid, decussation of pyramid and the ventral part of posterior funiculus in cervical, thoracic, lumbar and sacral segments of spinal cord. CONCLUSION: Luxol Fast Blue staining method may manifest the distribution of corticospinal tract with clear distinct in adult rats.

CHANGES OF VISUAL FINDINGS, ELECTRIC FEATURES AND STAINING OF AURICLES IN MALIGNANT TUMOR PATIENTS

In order to find a simple and conve-nient method of diagnosis and to probe intothe value of auricular diagnosis in malignanttumors so as to substantiate and

OBSERVATION OF MEGAKARYOCYTOPOIESIS IN HUMAN FETUS BY IMMUNOCYTOCHEMICAL STAINING WITH ANTI-PLATELET GLYCOPROTEIN ⅡB /ⅢA MONOCLONAL ANTIBODY

Fetal liver tissues obtained from 28 human fetuses with gestation age from 3 to 6 months and fetal bone marrow from 35 human fetuses from 3 to 7 months were observed by immunochemical staining with anti-platelet GPⅡ b / Ⅲa monoclonal antibody and ABC technique. In the fetal liver, megakaryocytes were wholly located among growing fetal liver cells and near foci of hemopoiesis. Some megakaryocytes in the fetal liver were small7890- lymphoid-like megakaryocytes. The size of megakaryocytes both in the fetal liver (14.79 ± 4.52μm) and in the fetal bone marrow (16.08±7.39 μm) was small, which did not vary significantly over the gestation age ranging from 3 to 6 or 7 months. However, the maturation stage of megakaryocytes in the fetal liver shifted to more mature stage with the advancement of gestation, although the maturation stage of megakaryocytes in the fetal bone marrow did not change with the advancement of gestation from 4 to 7 months, the megakaryocyte in the fetal bone marrow was less mature

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.

Immunohistochemical CD3 staining detects additional patients with celiac disease

AIM: To investigate whether performing immuno-histochemical CD3 staining, in order to improve the detection of intra-epithelial lymphocytosis, has an additional value in the histological diagnosis of celiac disease.METHODS: Biopsies obtained from 159 children were stained by hematoxylin and eosin(HE) and evaluated using the Marsh classification. CD3 staining was subsequently evaluated separately and independently.RESULTS: Differences in evaluation between the routine HE sections and CD3 staining were present in 20(12.6%) cases. In 10(6.3%) patients the diagnosis of celiac disease(Marsh Ⅱ and Ⅲ) changed on examination of CD3 staining: in 9 cases, celiac disease had initially been missed on the HE sections, while 1 patient had been over-diagnosed on the routine sections. In all patients, the final diagnosis based on CD3 staining, was concordant with serological results, which was not found previously. In the other 10(12.3%) patients, the detection of sole intra-epithelial lymphocytosis(Marsh Ⅰ) improved. Nine patients were found to have Marsh Ⅰ on CD3 sections, which had been missed on routine sections. Interestingly, the only patient with negative serology had Giardiasis. Finally, in 1 patient with negative serology, in whom Marsh Ⅰ was suspected on HE sections, this diagnosis was withdrawn after evaluation of the CD3 sections.CONCLUSION: Staining for CD3 has an additional value in the histological detection of celiac disease lesions, and CD3 staining should be performed when there is a discrepancy between serology and the diagnosis made on HE sections.

Fluorescence of Tb-Ibuprofenum-Phen Complex and New Tissue Staining Method

A new complex Tb(IB)_3(phen)·2H_2O that gives off green fluorescence was synthesized. The results show that the strong fluorescence emitting of 490 and 545 nm for Tb 3+ in the Tb(IB)_3(phen)·2H_2O complex is related to the transitions 5D_4-7F_6 and 5D_4-7F_5, respectively. The results indicate that the complex is concentrated on the nuclei regions in the section of onion toot tip tissues and the nuclei are high lighted under fluorescent microscope. It is possible that the Tb(IB)_3(phen)·2H_2O complex can be developed as a fluorescent dye in biological and pathological studies.

COMPARISON OF DIFFERENT STAINING METHODS FOR ANALYSING ESTERASE (EST) ISOZYME OF FUNGI AND PLANTS

EST isozymes are one of the frequently used biochemical markers in genetic analysis of fungi. and the staining is an important process in electrophoresis analysis of studying Est.The effects were compared ainong different stain recipes for Est of 3 kinds of fungi-Lentinus edodes. Pleurotus sapidus. Phellinus igriarius and 2 kinds of plants-Populus sp and Brassica chinensis. Of the four kinds of Est staining recipes tested.the recipe α-acetic acid-naphther showed the best effect.and followed by β-aceticacid-naphther, semicontent α-aceticacid-naphther and α+β-aceticacidnathpher.

Comparative Investigation of Alternative Negative Staining Reagents in Bacterial Morphological Study

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.

Experimental Study on the Effectiveness and Safety of Gardenia Blue Pigment on Anterior Capsule Staining

Objective: To explore the effect of gardenia blue pigment on the anterior capsule staining and its safety performance. Methods: In this study, New Zealand white rabbits were used as the research object to test the effect of gardenia blue pigment on the anterior capsule staining and anterior segment toxicity. In this study, gardenia blue stains of different concentrations (5%, 4%, 3%, 2%, 1%, 0.5%) were prepared to stain the anterior capsule of rabbit crystals, and the effect of the stain was observed to determine the optimal concentration of the stain. Twenty-seven healthy New Zealand white rabbits without eye disease were randomly divided into three groups: gardenia blue staining group;bio-blue positive drug group;physiological saline group. The intraocular pressure, anterior chamber inflammation, corneal endothelial injury, and anterior chamber angle pathological changes were measured in 1D, 7D, and 14D. Results: The results of this study showed that the effect of gardenia blue pigment on the anterior capsule of the lens was the best when the concentration of blue pigment was 2%. There was no change in intraocular pressure in the anterior chamber for a short period of time. There is no damage to the corneal endothelium. Conclusion: The results of this study show that the effect of gardenia blue pigment on the anterior capsule is best when the concentration of blue pigment is 2%, and the stain has no obvious toxic and side effects on the anterior segment of the eye.