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.

Tectona grandis (Teak Tree) Young Leaf Extract as a Histological Stain

Stains are applied to impart contrast to the tissue and identify particular features of interest. However, the use of synthetic dyes as staining reagents has been associated with significant human health challenges and pollution of the ecosystem. These developments have necessitated a shift towards using natural dyes that are eco-friendlier and readily available. We investigated the staining reaction patterns of teak tree leaves (Tectona grandis) dye extracts and explored their suitability as a cytoplasmic stain in micromorphological assessments. Dye extracts were prepared using acetone, methanol, and ethanol as solvents from air-dried (under shade) teak tree young leaves. The dye extracts were applied as a counterstain and evaluated against eosin in formalin-fixed paraffin-embedded (FFPE) bovine tissue sections at varying concentrations and different staining times. Teak tree leaves (Tectona grandis) dye extracts produced relatively varying staining intensities of reddish-brown cytoplasmic coloration when used on bovine tissue at different concentrations and staining times comparable to eosin and with blue-purple hematoxylin nuclear stain. The present study showed that Tectona grandis leaf dye extracts provide an excellent cytoplasmic staining pattern and can be used as an alternative counterstain in routine H&E staining techniques.

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.

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.

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.

Can the Prediction of Intrauterine Insemination Results by Used Aniline Blue Stain (ABS) and Sperm Chromatin Dispersion (SCD) Levels?

Introduction: This study aimed to perform routine seminal fluid analysis, sperm DNA fragmentation, and sperm function tests at the chromatin maturation level and evaluate pregnancy in the patients passing intrauterine insemination before starting Intrauterine Insemination (IUI) method. Materials and Methods: In this prospective study, 111 couples who underwent Intrauterine Insemination (IUI) in unexplained infertility patients were admitted to Al-Farah IVF and assisted reproductive center in Baghdad, Iraq between November 2020 and February 2021 were evaluated. Semen fluid analysis was performed based on (WHO 4th) guiding rules. In addition, Sperm Chromatin Dispersion (halo test) and sperm maturation were performed with Aniline Blue Stain (ABS). Results: Sperm Chromatin Dispersion (SCD) groups were compared in terms of pregnancy outcome;the positive pregnancy rate was found to be above in the normal SCD groups (p = 0.0005). In addition, Aniline Blue Stain (ABS) groups were compared in the terms of pregnancy outcome;the positive pregnancy rate was found to be higher in the normal ABS group (p = 0.017). Conclusion: Our study showed that the use of DNA fragmentation (SCD) and sperm maturation tests (ABS) together with routine semen analysis in intrauterine insemination cases will make a significant contribution to the prediction of Intrauterine Insemination (IUI) increased results. So, these results indicate a defect in the effect of DNA fragmentation on the outcome of intrauterine insemination.

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.

Advances in photodynamic therapy for port-wine stain and our experience

Port-wine stain(PWS)is a congenital capillary malformation that occurs in 0.3%–0.5%of newborns.The pulsed dye laser is the current gold standard treatment for PWS;however,its efficacy is poor.Photosensitizer photodynamic therapy(PDT)is considered a promising treatment for PWS.Here we provide a comprehensive overview of PDT.

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

选择是最可能的发展的卵母细胞是关键的为在 vitrofertilization 并且动物克隆。染色的灿烂的 cresyl 蓝色(BCB ) 在大型家畜被用于卵母细胞选举，但是它的更宽的用途需要推进评估。我们再划分进染色的那些(BCB+) 和那些的老鼠卵母细胞未沾污(BCB-) 根据他们的 ooplasm BCBcoloration。染色质配置，积云房间 apoptosis，细胞质的成熟和发展胜任在 BCB+ 和 BCB- 卵母细胞之间被比较。BCB+ 卵母细胞的胜任上的卵母细胞直径，性成熟和 gonadotropin 刺激的效果也被分析。在thelarge尺寸和中等尺寸的组，BCB+卵母细胞更大并且显示出更多的包围 nucleoli ( SN )染色质配置和早闭锁的更高的频率，并且他们也获得了更好细胞质的成熟(象细胞内部的 GSH 水平和 mitochondrialdistribution 的模式坚定)并且在试管内成熟( IVM )以后的更高发展的潜力比BCB卵母细胞。当没与 PMSG 告知时，成年老鼠比 prepubertal 老鼠与更高的胜任生产了更多的 BCB+ 卵母细胞。PMSG priming 增加了比例和 BCB+oocytes 的发展力量。在大尺寸的组的 BCB+ 卵母细胞在 medium-sizegroup 比他们的对应物显示出更多的 SN 染色质配置，更好细胞质的成熟和更高发展的潜力。染色的 BCB 能为卵母细胞选择被用作一个有效方法，这被结束，但是 BCB+ 卵母细胞的胜任可以与卵母细胞直径，动物性成熟和 gonadotropin 刺激变化。总起来说，这里描述的标准的系列将更好允许在为更好的开发选择卵母细胞的选择。

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.

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.

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: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. S. enteritidis was detected 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 was 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.

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.

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.

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.

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.

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.

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 stined,the simultaneous visualization of fluorochromatic 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 fluo rescent vital staining technique offered a useful means foridentification and selection of heterokaryons in gametoplast manipulation studies.