Rapid Staining Procedure Research Articles

P-041 Impact of cigarette smoking on level of sperm reactive oxygen species in normozoospermic men

Abstract Study question Does cigarette smoking influence the levels of sperm reactive oxygen species (ROS) in men with normal semen routine parameters? Summary answer Cigarettes impact the sperm ROS of normozoospermic smokers, whose sperm ROS level is elevated when they smoke equal to or more than 10 cigarettes daily. What is known already Increasing evidence indicates that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated ROS levels in semen. Cigarette smoking leads to an imbalance between ROS and antioxidants in the semen of smokers, which may negatively affect semen quality and male fertility. However, few studies concern whether cigarette smoking would influence the sperm ROS levels in normozoospermic men. Study design, size, duration Data of semen analysis from January 2021 to December 2023 were involved. Men with normal semen routine parameters, including sperm concentration, total sperm number, sperm progressive motility, total motility and normal forms of sperm morphology, were included in the study. Sperm ROS levels of normozoospermic men were retrospectively analyzed. Men with varicocele and leucocytospermia were excluded. Participants/materials, setting, methods Sperm count and motility were analyzed by the computer-assisted sperm analysis (CASA) system, while sperm morphology was examined after the Diff-Quik rapid staining procedure. Sperm ROS was measured by flow cytometry using the probe DCFH-DA. Sperm ROS levels of smokers and non-smokers were compared by the Mann-Whitney U test. The Kruskal-Wallis H test was used for the comparison of sperm ROS levels among the control group and smoking subgroups. Main results and the role of chance A total of 2946 subjects were involved in the present study. The included subjects were divided into smokers (n = 730) and non-smokers (n = 2216) according to the clinical records. And the included smokers were subsequently divided into three subgroups according to the daily cigarettes taken, which were group A (1∼9 cigarettes/day, n = 184), group B (1∼19 cigarettes/day, n = 315), and group C (>20 cigarettes/day, n = 231). No statistically significant differences in age or BMI were found between smokers and non-smokers with normozoospermia. The sperm ROS level of the smoker was higher than that of the non-smoker [14.53% (14.84%) vs. 13.07% (12.09%), p = 0.001]. Compared to the control group (non-smoker), the sperm ROS levels of group B [13.07% (12.09%) vs. 15.44% (14.28%), p = 0.002] and group C [13.07% (12.09%) vs. 14.80% (14.31%)., p = 0.042] were significantly elevated. ROS levels in the control group and group A were comparable [13.07% (12.09%) vs. 13.46% (15.74%)., p = 0.391]. Limitations, reasons for caution It is a single-center study and the above results were from a retrospective analysis. Wider implications of the findings The above results indicate that cigarette smoking, although it would not deteriorate semen routine parameters, may increase the ROS level in sperm. Elevated ROS in sperm may lead to an imbalance of redox in sperm, which may contribute to male infertility. Trial registration number not applicable

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Comparison of verdeluz orange G and modified Gallego stains

Tumors of the oral cavity include combinations of hard and soft tissues that may be difficult to identify using routine hematoxylin and eosin (H & E) staining. Although combination stains can demonstrate hard and soft tissues, trichrome stains, such as VanGieson and Masson, cannot differentiate dental hard tissues, such as dentin, cementum and osteoid. Modified Gallegos (MGS) and verdeluz orange G-acid fuchsin (VOF) stains can differentiate components of teeth. We used 10 tissue sections of decalcified bone and 10 pathologic tissue sections that contained different calcified tissues including peripheral ossifying fibroma, odontoma, central ossifying fibroma and cemento-ossifying fibroma. Sections were stained with H & E, VOF or MGS. H and E stained both hard tissues pink. VOF stained bone purple-red, cementum red and collagen blue. MGS stained bone green-blue, cementum red and collagen blue. VOF staining intensity and differentiation was better than MGS staining. VOF staining demonstrated hard tissue components distinctly and exhibited good contrast with the surrounding connective tissue. VOF also is a simple, single step, rapid staining procedure.

Staining Fission Yeast Filamentous Actin with Fluorescent Phalloidin Conjugates.

The Schizosaccharomyces pombe filamentous (F)-actin cytoskeleton drives cell growth, morphogenesis, endocytosis, and cytokinesis. The protocol described here reveals the distribution of F-actin in fixed cells through the use of fluorescently conjugated phalloidin. Simultaneous staining of cell wall landmarks (with calcofluor) and chromatin (with 4',6-diamidino-2-phenylindole, or DAPI) makes this rapid staining procedure highly effective for staging cell cycle progression, monitoring morphogenetic abnormalities, and assessing the impact of environmental and genetic changes on the integrity of the F-actin cytoskeleton.

Rapid protocol for visualization of rust fungi structures using fluorochrome Uvitex 2B.

BackgroundHistological examination using fluorochromes is one of the standard methods for observation of microorganisms in tissues and other compartments. In the study of fungi, especially those that cannot be cultured in axenic media such as biotrophic fungi, histological examination of processes associated with the fungal growth, differentiation, infection and other cellular functions can lead to the better understanding of host-parasite interactions. Fluorescence microscopy coupled with Fluorochrome Uvitex 2B have been extensively utilized to study rust fungi structures and host-pathogen interactions. In this study, we report development of a rapid staining protocol of the rust fungus Puccinia triticina using fluorochrome Uvitex 2B. The newly developed rapid procedure was compared with a standard staining technique to observe in planta fungal infection structures development during the wheat—Puccinia triticina interaction.ResultsWhile significantly reducing the time for staining, the rapid protocol described here was equally efficient or better compared to standard procedure in detecting fungal infection structures using Uvitex 2B. In the rapid staining procedure, pre-heating of the stain increased efficiency to detect all the infection structures including haustoria with highly reduced background noise from plant tissue.ConclusionThis staining process described here is simple and quick. It can be completed in 4 h, which is of 6 times faster than the standard Uvitex 2B staining procedure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0096-0) contains supplementary material, which is available to authorized users.

Staining Proteins in Gels

AbstractThis unit describes protocols for detecting proteins in SDS‐polyacrylamide gels. It describes methods for Coomassie blue and silver staining, as well as the fluorescent stains SYPRO Orange and Red. Staining with Coomassie blue is easier and more rapid; however, silver staining methods are considerably more sensitive and can thus be used to detect smaller amounts of protein. Alternative rapid staining procedures are provided for each method. Fluorescent staining is a popular alternative to the traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Curr. Protoc. Essential Lab. Tech. 6:7.4.1‐7.4.14. © 2012 by John Wiley & Sons, Inc.

Policies and Reporting Guidelines for Small Biopsy Specimens of Mediastinal Masses

目前，胸腺恶性肿瘤治疗方案大多是根据术 后病理确定，然而，多数临床治疗决策需要在术前 通过活检小标本的病理报告来制定。所以，术前活 检小标本的正确获取和病理解读对治疗决策的制定 显得非常重要[1]。这些标本包括细针活检标本，带 芯穿刺活检标本和手术切取活检标本[2-7]。由于胸 腺肿瘤的病理诊断对组织的获取方法和获取量都有较高 的要求，加之对病理的描述也没有统一的标准，使得小 标本在诊断胸腺瘤方面存在诸多问题。为此，ITMIG在 病理科医生和外科医生回顾相关文献和提出初步建议的 基础上，经集体讨论制定了活检规范操作流程，提出了 对纵隔肿物小活检标本处理和病理报告的建议。旨在为 术前患者的治疗提供一个统一和具有循证医学证据的方 法；同时，将有利于全球数据之间的比较和开展合作研 究，充分利用医疗资源。

Rapid Multiple Immunocytochemical Staining Method Using Microwave Irradiation for Intraoperative Cytology

To develop a more rapid and accurate staining procedure for intraoperative cytology through an assessment of a rapid multiple immunocytochemical staining method using microwave irradiation.A preliminary test of a triple immunostaining method using microwave irradiation was performed to determine optimal incubating conditions for primary antibodies against MOC-31, BerEP4 and carcinoembryonic antigen. The test samples were adenocarcinoma cells obtained by washings from 10 resected colorectal cancer specimens. Adenocarcinoma cells in peritoneal washings diagnosed by cytologic examination from 8 colorectal cancer patients were retrospectively examined using the previously determined optimal incubating conditions.High rates of positive cells (83-87%) with intense immunoreactions were obtained using a rapid process with primary antibody concentrations that were 2 times higher than those used for standard incubation at room temperature (82-86%). The staining under these conditions was completed within 19 minutes. Adenocarcinoma cells in the peritoneal washings were also intensely stained under these conditions.Using our microwave method, the processing time was dramatically shortened, and intense and sensitive immunoreactions were obtained. The present method is useful for the rapid and accurate diagnosis of intraoperative cytology.

A rapid dual staining procedure for the quantitative discrimination of prion amyloid from tissues reveals how interactions between amyloid and lipids in tissue homogenates may hinder the detection of prions

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases with no cure to this day, and are often associated with the accumulation of amyloid plaques in the brain and other tissues in affected individuals. The emergence of new variant Creutzfeldt–Jakob disease, an acquired TSE with a relatively long asymptomatic incubation period and unknown prevalence or incidence, which could potentially be iatrogenically transmitted, has prompted the need for sensitive and rapid methods of detection of the pathology indicator, the protease-resistant prion protein (PrP Sc), in tissues and on surgical instruments. To discriminate between common tissue proteins and amyloid-rich aggregates such as those formed by abnormal prion, we developed a quantitative thioflavin T/SYPRO Ruby dual staining procedure, used in combination with episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy for rapid scanning of samples. The detection limit of this direct observation technique applied to brain homogenates was greatly enhanced by the addition of Tween 20, as demonstrated in double-blind studies using various proportions of ME7-infected brain mixed with normal brain homogenate. The characteristic thioflavin T signal correlated with the relative amount of prion amyloid and proved at least 2-log more sensitive than the classic Western blot using the same prepared samples. This new sensitive microscopy procedure, which can be easily applied in instrument decontamination surveys, is likely to be more sensitive that Western blot in practice since it does not rely on the elution of resilient PrP Sc bound to the instrument surfaces. Our study also demonstrates how interactions between prion and lipid-rich tissue homogenates may reduce the sensitivity of such detection assays.

Evaluation of fast green FCF dye for non-lethal detection of integumental injuries in juvenile Chinook salmon Oncorhynchus tshawytscha

A rapid staining procedure for detection of recent skin and fin injuries was tested in juvenile Chinook salmon Oncorhynchus tshawytscha. Immersion of anesthetized fish for 1 min in aerated aqueous solutions of the synthetic food dye fast green FCF (Food Green 3) at concentrations of 0.1 to 0.5% produced consistent and visible staining of integumental injuries. A 0.1% fast green concentration was satisfactory for visual evaluation of injuries, whereas a 0.5% concentration was preferable for digital photography. A rinsing procedure comprised of two 30 s rinses in fresh water was most effective for removal of excess stain after exposure of fish. Survival studies in fresh water and seawater and histopathological analyses indicated that short exposures to aqueous solutions of fast green were non-toxic to juvenile Chinook salmon. In comparisons of the gross and microscopic appearance of fish exposed to fast green at various times after injury, the dye was observed only in areas of the body where epidermal disruption was present as determined by scanning electron microscopy. No dye was observed in areas where epidermal integrity had been restored. Further comparisons showed that fast green exposure produced more consistent and intense staining of skin injury sites than a previously published procedure using trypan blue. Because of its relatively low cost, ease of use and the rapid and specific staining of integumental injuries, fast green may find widespread application in fish health and surface injury evaluations.

Staining Proteins in Gels

AbstractThis unit describes protocols for detecting proteins in SDS‐polyacrylamide gels. It describes methods for Coomassie blue and silver staining, as well as the fluorescent stains SYPRO orange and red. Staining with Coomassie blue is easier and more rapid; however, silver staining methods are considerably more sensitive and can thus be used to detect smaller amounts of protein. Alternative rapid staining procedures are provided for each method. Fluorescent staining is a popular alternative to the traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining.

Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DAPI [4',6'-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and II) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 x 10(8) Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.

Staining Proteins in Gels

This unit describes protocols for detecting protein in a gel by either Coomassie blue, silver or fluorescent staining. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining (e.g., with SYPRO Orange or Red) is described as a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.

Staining Proteins in Gels

This unit describes protocols for detecting protein in a gel by either Coomassie blue staining or silver staining. The former is easier and more rapid; however, silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.

Background-free, fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes, zincon and ethyl violet.

A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1-1.5 h. The detection limit of this method is 8-15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes.

HLA-DQA1 allele typing by nonisotopic PCR-LIS-SSCP.

In the present study we used a simple and reliable method for HLA-DQA1 allele typing based on the single-stranded conformation polymorphism (SSCP) properties of DNA molecules obtained by PCR. The technique consists of PCR amplification of a DNA fragment comprising the second exon of the HLA-DQA1 gene, amplicon denaturation using a low ionic strength solution (LIS), and electrophoresis on a small native polyacrylamide gel, followed by a rapid silver staining procedure. In order to validate the technique and to obtain the allele patterns for the DQA1 gene, 50 cervical samples were typed using this methodology and the commercial Amplitype HLA DQA1 Amplification and Typing kit. All the alleles detected with the kit were characterized by the LIS-SSCP approach. This procedure proved to be useful for population screening and typing of the DQA1 gene as well as for detecting new alleles or mutations in the donor-recipient molecular matching of HLA class II genes.

Detection and initial characterization of novel capsular polysaccharide among diverse Campylobacter jejuni strains using alcian blue dye.

We have recently demonstrated that most strains of Campylobacter jejuni produce capsular polysaccharide (CPS), which can be detected by immunoblotting with homologous Penner antisera on polyvinylidene difluoride membranes (A. V. Karlyshev, D. Linton, N. A. Gregson, A. J. Lastovica, and B. W. Wren, Mol. Microbiol. 35:529-541, 2000). In this report, we describe a universal and rapid staining procedure using Alcian blue for C. jejuni CPS, which does not rely on the availability of antisera and identifies CPS in untypeable strains. Furthermore, Alcian blue staining identified CPS in its lipid-free form directly on Tricine gels, and we demonstrate that CPS is thermostable and is accumulated in the culture supernatant in a lipid-free form. The identification of a newly described CPS and its lipid-free form in C. jejuni should prove invaluable in studying the pathogenesis and epidemiology of this important pathogen.

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4—10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using α-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli β-glucuronidase activity using ELF-97 β-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution.

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.

A rapid staining procedure for arbuscules of living arbuscular mycorrhizas using neutral red as acidotropic dye.

Arbuscules are the most conspicuous structures of arbuscular mycorrhizas. Due to their large surface area, they are regarded as putative sites of nutrient exchange between host and symbiont. A new staining technique for arbuscules is presented in which arbuscules are selectively stained by acidotropic (i.e. based on the ion-trap mechanism) accumulation of neutral red (activity stain). Criteria to distinguish acidotropic staining from staining due to chemical affinity are presented together with approaches to minimize background staining and to check for complete penetration of the dye through the root tissues. The non-toxic staining technique allows detection of arbuscules in living roots within two to six hours. The histochemical properties of neutral red, microscopic techniques, potential pitfalls, the arbuscular life cycle, and aspects of future research are discussed.