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.

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.

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.

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.

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.

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.

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.

AUTOMATED ASSESSMENT OF EPIDERMAL THICKNESS AND VASCULAR DENSITY OF PORT WINE STAINS OCT IMAGE

Optical coherence tomography(OCT)enables in vivo imaging of port wine stains(PWS)lesions.The knowledge of vascular struct ure and epidermal thickness(ET)of PWS may aid the objectivediagnosis and optimal treatment.To obtain the structural parameters more rapidly and avoiduser intervention,an automated algorithm of energy map is introduced based on intensity andedge information to extract the skin surface using dynamic programming method.Subsequently,an averaged A-scan analysis is performed to obtain the mean ET and the relative intensity ofdermis indicating the corresponding vascular density.This approach is currently successfullyapplied in clinical diagnosis and shows promising guidance and assessment of PDT treatment.

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.

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

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.

DNA DIFFERENTIAL STAINING AND ITS CLINICAL APPLICATION

DNA differential stain is a simple method distinguishing cells of proliferative from quiescent stage. Double stranded DNA in quiescent cells is easily denatured by weak acid into single strand. As double stranded nucleic acid combined with methyl green and single stranded nucleic acid with pyronin, we make use of methyl green pyronin staining method to the cells treated with weak acid to distinguish proliferating from quiescent cells. This paper reports the observation of leukemia cells in the bone marrow smears of 100 cases of untreated acute leukemia by DNA differential staining method. The percentage of Go cells was lowest in ALL and highest in APL.

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.

Evaluation of Fluorescent Stains for Viability Assessment of the Potato Cyst Nematodes <i>Globodera pallida</i>and <i>G. ellingtonae</i>

Potato cyst nematodes (PCNs), Globodera pallida and Globodera rostochiensis, are quarantine pests of potato which cause significant damage to production and farm gate revenue worldwide. Accurately assessing viability of PCN eggs is important for eradication and management programs. The goal of this study was to develop a quick and reliable fluorescent staining method to evaluate viability of G. pallida and Globodera ellingtonae eggs. The staining efficiency of eight fluorescent stains was evaluated using G. pallida eggs compared with the conventional Meldola’s Blue (MB) staining method. The staining efficiency of the fluorescent stains ranged from 80.33 ± 2.99 (Sytox Green) to 100% (Acridine Orange) for non-viable eggs. Two stains were further evaluated for their efficiency in assessing viability of encysted eggs from five different greenhouse-reared G. pallida cyst sources which contained both viable and non-viable eggs. For the G. pallida cyst sources, viability ofencysted eggs were estimated to be 41.02 ± 3.81 to 62.66% ± 3.12% when stained with Acridine Orange (AO) and 79.52% ± 1.54% viability for G. ellingtonae. Both staining time and stain concentration were significant for staining efficiency of released and encysted eggs. Staining time and concentration were optimized for released eggs at 4 h at 10 μg/ml and for encysted eggs at 16 h at 25 μg/ml respectively for AO. Fluorescent stains accurately and rapidly assessed percent egg viability and were determined to be as sensitive as a seven-day incubation with the Meldola’s Blue staining method.

Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining

By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.

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.

Diagnostic Performance of glmM Gene and Histological Stains for Detection of Helicobacter pylori in Gastric Biopsy from Patients Admitted to Wad Madani Teaching Hospital, Sudan

Helicobacter pylori is the microbial agent most responsible for gastro-duodenal ulcer and chronic gastritis, which can develop into carcinoma of the stomach. This study was performed in Wad Medani Teaching Hospital, Sudan to detect Helicobacter pylori in stomach samples, and evaluate the performance of the tests used, which were histological stains and PCR. Gastric biopsies were obtained from 105 referred patients during endoscopy, and fixed specimens examined by haematoxylin-eosin and Warthin-Starry silver stains, while DNA was extracted for glmM gene amplification. Epigastric pain was the most common symptom at 78% (82/105) and chronic gastritis recorded with 71% (68/105) of endoscopy results. Warthin-Starry silver stain gave 31% (33/105) as positive for Helicobacter pylori followed by glmM gene 27% (28/105) and haematoxylin-eosin 24% (25/105). The study indicated good performance of histological staining and high specificity of glmM gene in detection of Helicobacter pylori from gastric biopsies.

The superiority and feasibility of 2,3,5-triphenyltetrazolium chloride-stained brain tissues for molecular biology experiments based on microglial properties

Background:TTC(2,3,5-triphenyltetrazolium chloride)staining is the most commonly used method in identifying and assessing cerebral infarct volumes in the transient middle cerebral artery occlusion model.Given that microglia exhibit different morphologies in different regions after ischemic stroke,we demonstrate the superiority and necessity of using TTC-stained brain tissue to analyze the expression of various proteins or genes in different regions based on microglia character.Methods:We compared brain tissue(left for 10 min on ice)from the improved TTC staining method with penumbra from the traditional sampling method.We identified the feasibility and necessity of the improved staining method using real time(RT)-PCR,Western blot,and immunofluorescence analysis.Results:There was no protein and RNA degradation in the TTC-stained brain tissue group.However,the TREM2 specifically expressed on the microglia showed a significant difference between two groups in the penumbra region.Conclusions:TTC-stained brain tissue can be used for molecular biology experiments without any restrictions.In addition,TTC-stained brain tissue shows greater superiority due to its precise positioning.

Introducing a Rapid and Safe Method for Myeloperoxidase Staining

Background: Myeloperoxidase staining is used to differentiate leukemias since several decades. Despite implementation of flow cytometric, cytogenetic and molecular techniques for identification of leukemic blasts, histochemical stains such as myeloperoxidase stain are persistently used for better classification of leukemias. The myeloperoxidase staining is a time consuming and hazardous procedure. The present report describes a sensitive, rapid and easy method for assessment of peroxidase activity. Materials and Methods: Bone marrow aspiration slides were stained with Dako product: Code number: K3467 containing DAB chromogen (3,3-diaminobenzidine in chromogen solution) and substrate buffer (Imidasole-HCL buffer, PH 7.5 containing hydrogen peroxide and an anti microbial agent) in a rapid procedure taking only ten minutes time. The staining needs no material preparation steps. Neutrophils in the slide are taken as positive control or another normal smear was costained to be used as control. All cases were followed up with flow cytometry and cytogenetic studies. Result: The reaction product of this stain is brown and granular. Promyelocytes and myelocytes are the most strongly staining cells with positive (primary) granules. Lymphoblasts are negative. The result of classification of leukemias with this technique was in concordance with flow cytometric immunophenotyping. Discussion: Many practical techniques have been described using benzidine as an indicator for myeloperoxidase staining. Benzidine is a carcinogenic material and its usage is severely restricted in laboratory. Formerly we prepared requisite materials for myeloperoxidase staining by hazardous ways (boiling), but we decided to apply ready to use 3,3-diaminobenzidine (DAB), which is used in final step of immunohistochemistry stains. Conclusion: Use of 3,3-diaminobenzidine (DAB) is highly recommended for myeloperoxidase staining, while the result is extraordinary and fully compatible with flow cytometry and the method is safe and rapid.

Reduction and Block Staining of Human Hair Shafts and Insect Cuticles by Ammonium Thioglycolate to Enhance Transmission Electron Microscopic Observations

We have developed a method where, after glutaraldehyde fixation, human hair shafts and insect cuticles are treated with ammonium thioglycolate (ATG) to improve ultrastructural staining. Conventional transmission electron microscopic (TEM) preparations do not distinguish the A-layer and the exocuticles of hair shafts. However, after ATG treatment, the A-layer appears in higher contrast. ATG treatment has also been used to observe the fibrillar structure in the cortex. In the cuticle of beetles, the epicuticle is stained by ATG. Although the human hair shaft (keratin) and insect cuticle (chitin) are composed of different materials, both can be reduced by the ATG solution. The ammonium in the ATG solution reacts with hair shafts and insect cuticles, causing a reduction of swelled cuticles. Therefore, ATG not only stains, but also reduces human hair shafts and the cuticles of beetles.