Staining Protocol Research Articles

Automated Quantification of Tertiary Lymphoid Structures in Human Tumor Samples Using Immunofluorescence and AI-Powered Analysis Pipeline.

The tumor microenvironment (TME) is a complex entity comprising not only tumor cells but also immune, stromal, and endothelial cells. Preclinical and clinical studies indicate that the density, localization, function, and organization of immune infiltration can influence survival probability and treatment response in many cancers. Among these cell organizations, the clustering of T and B cells into tertiary lymphoid structures (TLS) has been associated with favorable clinical outcomes. In this protocol, we propose a protocol for 7-plex immunofluorescence staining for identifying six cell types enriched in TLS in formalin-fixed paraffin-embedded (FFPE) human tumor sections. Additionally, we provide a detailed methodology for quantifying these cell subtypes using the Halo-AI analysis software. This approach will enable a more precise and detailed characterization of TLS in the TME, opening new avenues for understanding their role in anticancer treatment response.

Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure.

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying and visualizing tissue antigens. However, traditional IHC staining methods have limitations in distinguishing various subtypes of immune cells. This challenge has driven scientists to explore new technologies and methodologies for precise identification and differentiation of immune cell subtypes. In recent years, multiplex IHC has emerged as a solution, enabling the simultaneous detection of multiple antigens and their visualization within the same tissue sample. Uterine natural killer (uNK) cells play a pivotal role in early pregnancy processes, including decidualization, remodeling of uterine spiral arteries, and embryo implantation. Different subtypes of uNK cells exhibit different functions, allowing them to coordinate various biological events for successful embryo development and pregnancy. Therefore, in-depth research on uNK cell subtypes is essential for elucidating immune regulation mechanisms during pregnancy. Such studies provide valuable insights and novel approaches for addressing related conditions such as infertility and recurrent reproductive failure. This paper introduces a detailed multiplex IHC staining protocol for studying the density of four subtypes of uNK cells in endometrial specimens during the window of implantation (WOI). The protocol includes sample preparation, optimization of subtype markers, microscopic imaging, and data analyses. This multiplex IHC staining protocol offers high specificity and sensitivity, enabling simultaneous detection of different uNK cell subtypes, thus providing researchers with a powerful tool to explore the intricacies and mechanisms of immune regulation during pregnancy.

A refined TTC assay precisely detects cardiac injury and cellular viability in the infarcted mouse heart

Histological analysis with 2,3,5-triphenyltetrazolium chloride (TTC) staining is the most frequently used tool to detect myocardial ischemia/reperfusion injury. However, its practicality is often challenged by poor image quality in gross histology, leading to an equivocal infarct-boundary delineation and potentially compromised measurement accuracy. Here, we introduce several crucial refinements in staining protocol and sample processing, which enable TTC images to be analyzed with light microscopy. The refined protocol involves a two-step TTC staining process (perfusion and immersion) and subsequent Zamboni fixation to differentiate myocardial viability and necrosis, and use of Coomassie brilliant blue to label area-at-risk. After the duo-staining steps were completed, the heart sample was embedded and sliced transversally by a cryostat into a series of thin sections (50 µm) for microscopic analysis. The refined TTC (redTTC) assay yielded remarkably high-quality images with striking color intensity and sharply defined boundaries, permitting unambiguous and reliable delineation of the infarct and area-at-risk. In the same animals, the redTTC assay showed good agreement with the in-vivo gold standard measurements (LGE and MEMRI). Meanwhile, redTTC imaging allows tracking of viable cardiomyocytes at cellular resolution, and with this enhanced capability, we convincingly demonstrated the pro-survival action of stem cells based-therapy. Therefore, the redTTC assay represents a significant technical advance that permits precise detection of the true extent of cardiac injury and cardiomyocyte viability. This approach is cost-effective and may be adapted for use in diverse applications, making it highly appealing to many laboratories performing ischemia/reperfusion injury experiments.

Artificial intelligence can be trained to predict c-KIT-11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides.

Numerous prognostic factors are currently assessed histologically and immunohistochemically in canine mast cell tumors (MCTs) to evaluate clinical behavior. In addition, polymerase chain reaction (PCR) is often performed to detect internal tandem duplication (ITD) mutations in exon 11 of the c-KIT gene (c-KIT-11-ITD) to predict the therapeutic response to tyrosine kinase inhibitors. This project aimed at training deep learning models (DLMs) to identify MCTs with c-KIT-11-ITD solely based on morphology. Hematoxylin and eosin (HE) stained slides of 368 cutaneous, subcutaneous, and mucocutaneous MCTs (195 with ITD and 173 without) were stained consecutively in 2 different laboratories and scanned with 3 different slide scanners. This resulted in 6 data sets (stain-scanner variations representing diagnostic institutions) of whole-slide images. DLMs were trained with single and mixed data sets and their performances were assessed under stain-scanner variations (domain shifts). The DLM correctly classified HE slides according to their c-KIT-11-ITD status in up to 87% of cases with a 0.90 sensitivity and a 0.83 specificity. A relevant performance drop could be observed when the stain-scanner combination of training and test data set differed. Multi-institutional data sets improved the average accuracy but did not reach the maximum accuracy of algorithms trained and tested on the same stain-scanner variant (ie, intra-institutional). In summary, DLM-based morphological examination can predict c-KIT-11-ITD with high accuracy in canine MCTs in HE slides. However, staining protocol and scanner type influence accuracy. Larger data sets of scans from different laboratories and scanners may lead to more robust DLMs to identify c-KIT mutations in HE slides.

A Novel Monoclonal Antibody Against a Modified Vaccinia Ankara (MVA) Envelope Protein as a Tool for MVA Virus Titration by Flow Cytometry.

Modified vaccinia Ankara (MVA) virus is a widely used vaccine platform, making accurate titration essential for vaccination studies. However, the current plaque forming unit (PFU) assay, the standard for MVA titration, is prone to observer bias and other limitations that affect accuracy and precision. To address these challenges, we developed a new flow cytometry-based quantification method using a highly specific monoclonal antibody (mAb) for the detection of MVA-infected cells, as a more accurate titration assay. Through previous work, we serendipitously identified three MVA-specific hybridoma antibody clones, which we characterized through ELISA, immunoblot, and flow cytometry, confirming their specificity for MVA. Sequencing confirmed that each antibody was monoclonal, and mass spectrometry results revealed that all mAbs target the MVA cell surface binding protein (CSBP, MVA105L). We next optimized the titration protocol using the most effective mAb, 33C7 by refining culture conditions and staining protocols to enhance sensitivity and minimize background. Our optimized method demonstrated superior sensitivity, reliability, and reduced processing time when compared with the traditional PFU assay, establishing it as a more accurate and efficient approach for MVA titration.

Performance of Gram Stain Characteristics for Predicting Culture Growth in Lower Respiratory Specimens Across Multiple Institutions

Abstract Data from Gram stains performed on clinical specimens can guide clinical decision-making and downstream laboratory workflows. However, the extent to which Gram stain results predict gold-standard culture results, and the generalizability of this performance across sites, warrants additional exploration. We sought to discover which Gram stain features would be associated with clinically significant culture growth (“growth”). To do so, 574 remnant specimens from the lower respiratory tract collected from four institutions were analyzed. A standardized Gram stain and culture protocol was performed across all study sites. Final reference organism identification was assigned using MALDI-ToF MS, and clinically significant growth was defined as any organism reported to the medical record. Gram stains with mixed microorganisms detected were considered concordant if ≥2 organisms of different Gram stain appearances were identified in culture. Growth was observed in 53% of specimens, including 25% of specimens in which Gram stain demonstrated no organisms. The 95% confidence intervals (CI) of the site-wise growth rates were 30-42%, 42-52%, 51-67%, and 60-77% (p < 0.001 by Chi-squared). By Gram stain morphology, 53-69% of specimens with Gram-positive cocci grew any clinically significant bacteria, while 31-49% grew a concordant Gram-positive cocci. 65-85% of specimens with Gram-negative bacilli grew any clinically significant bacteria, while 59-74% grew a concordant Gram-negative bacilli. 63-83% of specimens with mixed microorganisms grew 2 or more clinically relevant bacteria of differing Gram stain morphologies. Specimens from bronchoalveolar lavages (BALs) were less likely to have growth (30-42%) than endotracheal aspirates (66-77%). Polymorphonuclear leukocyte (PMNs) abundance was associated with culture growth, with Gram stains showing no, rare, few, moderate, or abundant PMNs demonstrating growth 28, 44, 52, 60, and 67% of the time (p < 0.001). Specimens with None and few squamous epithelial cells on Gram stain demonstrated growth 38-52% and 59-68% of the time (p < 0.001). No significant difference in growth was observed for red blood cells. Organism abundance on Gram stain was significantly associated with culture growth, with growth rate CIs being 20-32% for no organisms, 44-62% for rare, 58-77% for few, 70-93% for moderate, and 81-95% for abundant organisms seen. The extent to which these associations could guide the development of decision rules or machine learning models that demonstrate acceptable negative predictive value for pathogen growth to alleviate the operational burden of low-utility cultures is an exciting future direction to explore.

The Power of Reagent Titration in Flow Cytometry.

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration-response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution.

A Modified Bleaching Method for Multiplex Immunofluorescence Staining of FFPE Tissue Sections.

Multiplex immunofluorescence (mIF) staining plays an important role in profiling biomarkers and allows investigation of co-relationships between multiple biomarkers in the same tissue section. The Cell DIVE mIF platform (Leica Microsystems) employs an alkaline solution of hydrogen peroxide as a fluorophore inactivation reagent in the sequential staining, imaging, and bleaching protocol for use on FFPE sections. Suboptimal bleaching efficiency, degradation of tissue structure, and loss of antigen immunogenicity occasionally are encountered with the standard bleaching process. To overcome these impediments, we adopted a modified photochemical bleaching method, which utilizes an intense LED light exposure concurrent with the application of hydrogen peroxide. Repeated stain/bleach rounds with different antibodies were performed on breast tissue and other tissue sections. Residual signal after conventional bleaching and the modified technique were compared and tissue integrity and antigen immunogenicity were assessed. The modified technique effectively eliminates fluorescence signal from previous staining rounds and produces consistent results for multiple rounds of staining and imaging. With the modified method, photochemical treatments did not destroy tissue sub-cellular contents, and the tissue antigenicity was well preserved during the entire mIF process. Overall processing time was reduced from 36 to 30 hours in an mIF procedure with 8 rounds. With the conventional method, tissue quality was highly degraded after 8 rounds. The new technique allows reduced turn-around time, provides reliable fluorophore removal in mIF with excellent maintenance of tissue integrity, facilitating studies of the co-localization of multiple biomarkers in tissues of interest.

The Buds of Oscarella lobularis (Porifera, Homoscleromorpha): A New Convenient Model for Sponge Cell and Evolutionary Developmental Biology.

The comparative study of the four non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, and Porifera) provides insights into the origin of bilaterian traits. To complete our knowledge of the cell biology and development of these animals, additional non-bilaterian models are needed. Given the developmental, histological, ecological, and genomic differences between the four sponge classes (Demospongiae, Calcarea, Homoscleromorpha, and Hexactinellida), we have been developing the Oscarella lobularis (Porifera, class Homoscleromorpha) model over the past 15 years. Here, we report a new step forward by inducing, producing, and maintaining in vitro thousands of clonal buds that now make possible various downstream applications. This study provides a full description of bud morphology, physiology, cells and tissues, from their formation to their development into juveniles, using adapted cell staining protocols. In addition, we show that buds have outstanding capabilities of regeneration after being injured and of re-epithelization after complete cell dissociation. Altogether, Oscarella buds constitute a relevant all-in-one sponge model to access a large set of biological processes, including somatic morphogenesis, epithelial morphogenesis, cell fate, body axes formation, nutrition, contraction, ciliary beating, and respiration.

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of InVitro Cultivation and Membrane Integrity Methods.

Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%. Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

KMnO4/Pb staining allows uranium free imaging of tissue architectures in low vacuum scanning electron microscopy

Scanning electron microscopy under low-vacuum conditions allows high-resolution imaging of complex cell/tissue architectures in nonconductive specimens. However, the conventional methods for metal staining of biological specimens require harmful uranium compounds, which hampers the applications of electron microscopy. Here, we introduce a uranium-free KMnO4/Pb metal staining protocol that allows multiscale imaging of extensive cell/tissue architectures to intensive subcellular ultrastructures. The obtained image contrast was equivalent to that of Ur/Pb staining and sufficient for ultrastructural observation, showing the fine processes of podocytes in the glomerulus, which were invisible by light microscopy. The stainability in the elastic tissue indicated that the distinct histochemical properties of KMnO4 oxidation led to Pb deposition and BSE signal enhancement superior to Ur staining. Elemental analysis clarified that the determinant of the backscattered electron signal intensity was the amount of Pb deposition enhanced by KMnO4 oxidation. This user-friendly method is anticipated to create a new approach for biomedical electron microscopy.

Fast Green FCF Improves Depiction of Extracellular Matrix in Ex Vivo Fluorescence Confocal Microscopy.

Rapid microscopic analysis of tissue is an essential diagnostic tool in oncological surgery. The gold standard for intraoperative histological tissue evaluation is frozen sections. However, frozen sections are prone to a variety of artefacts and require skilled staff and specialized lab equipment. A potential method for rapid intraoperative tissue evaluation that does not require fixation, freezing, or sectioning of the tissue is ex vivo fluorescence confocal microscopy (FCM). The visualization of the structurally important extracellular matrix (ECM) in conventional ex vivo FCM lags behind the standards of conventional histology. The objective of this study was to find a stain that would improve the depiction of the ECM to resemble FFPE H&E sections as closely as possible. Eleven different tissue stains were tested on 122 tissue samples submitted to the Department of Pathology at the Medical University of Vienna. This study was conducted on the RS-G4 Upright (Caliber I.D. Rochester, NY, USA, distributed in Europe by MAVIG GmbH, Munich, Germany). Fast Green FCF (FGFCF) in combination with acridine orange as a nuclear stain improved the visibility of the structural details of the ECM. Morphological details in FCM were equivalent or even superior to frozen sections in most analyzed categories. The addition of FGFCF to the conventional staining protocol improves the assessment of the ECM and analysis of fibrosis. The rapid staining protocol is compatible with an application in intraoperative microscopy.

An initial game-theoretic assessment of enhanced tissue preparation and imaging protocols for improved deep learning inference of spatial transcriptomics from tissue morphology.

The application of deep learning to spatial transcriptomics (ST) can reveal relationships between gene expression and tissue architecture. Prior work has demonstrated that inferring gene expression from tissue histomorphology can discern these spatial molecular markers to enable population scale studies, reducing the fiscal barriers associated with large-scale spatial profiling. However, while most improvements in algorithmic performance have focused on improving model architectures, little is known about how the quality of tissue preparation and imaging can affect deep learning model training for spatial inference from morphology and its potential for widespread clinical adoption. Prior studies for ST inference from histology typically utilize manually stained frozen sections with imaging on non-clinical grade scanners. Training such models on ST cohorts is also costly. We hypothesize that adopting tissue processing and imaging practices that mirror standards for clinical implementation (permanent sections, automated tissue staining, and clinical grade scanning) can significantly improve model performance. An enhanced specimen processing and imaging protocol was developed for deep learning-based ST inference from morphology. This protocol featured the Visium CytAssist assay to permit automated hematoxylin and eosin staining (e.g. Leica Bond), 40×-resolution imaging, and joining of multiple patients' tissue sections per capture area prior to ST profiling. Using a cohort of 13 pathologic T Stage-III stage colorectal cancer patients, we compared the performance of models trained on slide prepared using enhanced versus traditional (i.e. manual staining and low-resolution imaging) protocols. Leveraging Inceptionv3 neural networks, we predicted gene expression across serial, histologically-matched tissue sections using whole slide images (WSI) from both protocols. The data Shapley was used to quantify and compare marginal performance gains on a patient-by-patient basis attributed to using the enhanced protocol versus the actual costs of spatial profiling. Findings indicate that training and validating on WSI acquired through the enhanced protocol as opposed to the traditional method resulted in improved performance at lower fiscal cost. In the realm of ST, the enhancement of deep learning architectures frequently captures the spotlight; however, the significance of specimen processing and imaging is often understated. This research, informed through a game-theoretic lens, underscores the substantial impact that specimen preparation/imaging can have on spatial transcriptomic inference from morphology. It is essential to integrate such optimized processing protocols to facilitate the identification of prognostic markers at a larger scale.

Effect of zinc oxide-eugenol endodontic paste on planktonic aggregates and biofilm of Enterococcus faecalis - An atomic force microscopy evaluation

ObjectiveThis work aimed to evaluate the in vitro effect of zinc oxide-eugenol paste (ZOE) on planktonic aggregates (EfPA) and biofilm (EfBio) of Enterococcus faecalis, focusing on their morphological aspects observed and analyzed using atomic force microscopy (AFM). DesignThe eugenol and paste were characterized by Gas Chromatography coupled with Mass Spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FTIR), respectively. The effect of ZOE on EfPA and EfBio was evaluated by a direct-contact test through colony counting and crystal violet staining protocol. AFM images of untreated and treated EfPA and EfBio growth on bovine dentin were obtained to analyze the morphological damage caused by the treatments. ResultsThe characterization showed high purity in the eugenol composition and chemical interaction between the components of the paste. A bactericidal effect on aggregates was observed after 6 h of exposure, and on biofilm after 24 h of treatment (p < 0.001). A disruptive effect on the biofilm was also evident. AFM images revealed the formation of EfPA, with a notable presence of an exopolysaccharide matrix. After 6 h of ZOE treatment, there was a significant increase in the size and surface roughness profile of treated cells (p < 0.05). Loss of typical cell morphology was observed after 24 h. The effect on the biofilm showed a tendency towards a less condensed biofilm pattern in the treated group, with no differences in surface roughness. ConclusionZOE presents bactericidal action on EfPA and EfBio, promoting significant morphological changes after treatment, especially in the aggregates.

Quantitative and comparative assessment of dyes and protocols for rapid ex vivo microscopy of fresh tissues

Using ex vivo microscopy, virtual pathology can improve histological procedures by providing pathology images in near real-time without tissue destruction. Several emerging and promising approaches leverage fast-acting small-molecule fluorescent stains to replicate traditional pathology structural contrast, combined with rapid optical sectioning microscopes. However, several vital challenges must be addressed to translate virtual pathology into the clinical environment. One such challenge is selecting robust, reliable, and repeatable staining protocols that can be adopted across institutions. In this work, we addressed the effects of dye selection and staining protocol on image quality in rapid point-of-care imaging settings. For this purpose, we used structured illumination microscopy to evaluate fluorescent dyes currently used in the field of ex vivo virtual pathology, in particular, studying the effects of staining protocol and temporal and photostability on image quality. We observed that DRAQ5 and SYBR gold provide higher image quality than TO-PRO3 and RedDot1 in the nuclear channel and Eosin Y515 in the extracellular/cytoplasmic channel than Atto488. Further, we found that TO-PRO3 and Eosin Y515 are less photostable than other dyes. Finally, we identify the optimal staining protocol for each dye and demonstrate pan-species generalizability.

The effect of aging on mast cell density in human skin: a comparative analysis of photoexposed and photoprotected regions.

Mast cells are mononuclear cells originating from bone marrow. They produce various biologically active substances, which allow them to actively participate in immune and inflammatory processes associated with intrinsic and extrinsic skin aging. This research focused on distribution and density of mast cells in healthy skin in different stages of skin aging. This project included samples of photoexposed and photoprotected skin, obtained from 90 cadavers aged 0-82 years. The samples were classified into five age groups: newborns, young age, middle age, senior age and the oldest age. In order to visualize the mast cells, we have employed several histochemical staining protocols. The number of mast cells of the photoexposed skin significantly correlated to the individual's age. The number of mast cells of the photoprotected skin was in general statistically significantly lower in younger compared to older groups; however, the correlation of the mast cell density in photoprotected skin and the age did not reach statistical significance. In middle age, senior age and the oldest age groups, a significantly higher number of mast cells was recorded in the skin of the photoexposed compared to photoprotected region. The increase in mast cell density correlated with age only in photoexposed skin. Age-related higher accumulation of dermal mast cells in photoexposed skin can be an important factor in the photoaging process, as well as the contributing factor in the occurrence of skin cancer.

Color Stability Of Various Resin Sealants After Staining Protocol

Color Stability Of Various Resin Sealants After Staining Protocol

Standardization of CD30 immunohistochemistry staining among three automated immunostaining platforms.

The identification of CD30 expression by immunohistochemistry is essential for the treatment of lymphomas using an antibody-drug conjugate targeting CD30. However, no standardized protocol for CD30 staining has been available. In this study, we compared three common automated immunostaining platforms {Bond III (B III), Dako Omnis (DO) and Ventana BenchMark ULTRA (VBMU)}. A primary antibody for CD30, the Ber-H2 clone, was diluted 50- to 400-fold for B III and DO, and ready-to-use antibody was used for VBMU. An enhancement step using a linker was introduced in all protocols. First, several candidate dilutions were selected for each platform by staining six cases. These candidate conditions were then confirmed with 60 cases of various types of peripheral T-cell lymphomas (PTCLs). The concordance rates of CD30 expression among platforms differed depending on cutoff values and antibody dilutions, except for anaplastic large cell lymphoma. The concordance rates among three platforms in the evaluation of "positive" or "negative" were 100% and 97% when the cutoff values were 1% and 10% respectively, if using 400-diluted antibody in B III and 100-diluted antibody in DO. This study demonstrated the feasibility of equalizing CD30 staining of PTCLs among different platforms by adjusting protocols.

Protocol for the quantitative analysis of images retrieved by multiplex immunofluorescence staining to allow cell type-specific spatial phenotyping of markers of interest in the human placenta

In contrast to other tissues, the placenta consists of numerous functionally different cell types, distributed in a markedly dissimilar manner within one placenta and between different cases. To evaluate pathology-specific changes in cell phenotype and expression of molecular markers it is important to establish a multi staining method combining immunohistological identification of the cell type with staining of proteins of interest. We successfully established a protocol for a 6-plex antibody panel for multiplex immunofluorescence. Here, we report the staining protocol and the establishment of the quantification algorithm we developed.

Application of a xenopericardial plate for closing a laparostomy in conditions of a purulent-inflammatory process

The aim of the study is to study the morphological changes in tissue in the laparostomy area when it is closed with a xenopericardial plate under conditions of an active purulent-inflammatory process. Material and methods. Analysis of data from patients at the Penza regional clinical hospital named after. N.N. Burdenko, with diseases of the abdominal organs, complicated by diffuse purulent peritonitis. During treatment, xenopericardial plates with perforations in a checkerboard pattern were used (hole diameter 0.5 cm). Disinfection of the material was carried out with a sterile solution of furacilin. Using the standard hematoxylin and eosin staining protocol, as well as alternative staining methods according to Van Gieson and Weigert, light-optical examination of stained sections was carried out at a magnification of 40–400 times. Results. In 8 out of 9 patients participating in the study, a favorable course of the inflammatory process was observed; on the 21st day after laparotomy, the symptoms of peritonitis were eliminated, the surgical wound was sutured. In one case, progression of local and general signs of peritonitis was observed within two weeks. After replacing the xenopericardial plate, the course of changes in the surgical wound was favorable. On day 21, the laparotomy wound was also sutured. Repeated examination of the biomaterial one year and two months after implantation of the xenopericardial plate into the anterior abdominal wall did not reveal signs of inflammation. Conclusions. The xenopericardium performed well in conditions of purulent inflammation and in most cases was not subject to melting. In the long term, biointegration of the xenopericardium into its own connective tissue occurred.