Image Analysis Protocol for DNA/RNA/Immunofluorescence (IF)-seqFISH Data.

Imaging-based spatial multi-omics technologies facilitate the analysis of higher-order genomic structures, gene transcription, and the localization of proteins and posttranslational modifications (PTMs) at the single-allele level, thereby enabling detailed observations of biological phenomena, including transcription machinery within cells and tissues. This chapter details the principles of such technologies, with a focus on DNA/RNA/immunofluorescence (IF) sequential fluorescence in situ hybridization (seqFISH). A comprehensive step-by-step protocol for image analysis is provided, covering image preprocessing, spot detection, and data visualization. For practical application, complete Jupyter Notebook codes are made available on GitHub ( https://github.com/Ochiai-Lab/seqFISH_analysis ).

A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence.

Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

PUPAID: A R + ImageJ pipeline for thorough and semi-automated processing and analysis of multi-channel immunofluorescence data.

PUPAID is a workflow written in R + ImageJ languages which is dedicated to the semi-automated processing and analysis of multi-channel immunofluorescence data. The workflow is designed to extract fluorescence signals within automatically-segmented cells, defined here as Areas of Interest (AOI), on whole multi-layer slides (or eventually cropped sections of them), defined here as Regions of Interest (ROI), in a simple and understandable yet thorough manner. The included (but facultative) R Shiny-based interactive application makes PUPAID also suitable for scientists who are not fluent with R programming. Furthermore, we show that PUPAID identifies significantly more cells, especially in high-density regions, as compared to already published state-of-the-art methods such as StarDist or Cellpose. For extended possibilities and downstream compatibility, single cell information is exported as FCS files (the standardized file format for single cell-based cytometry data) in order to be openable using any third-party cytometry analysis software or any analysis workflow which takes FCS files as input.

A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence.

Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Development of an automated high-content immunofluorescence assay of pSmads quantification: Proof-of-concept with drugs inhibiting the BMP/TGF-ß pathways.

INTRODUCTION: Bone morphogenetic proteins (BMPs) and transforming growth factors (TGF-ß) are members of the TGF-ß superfamily, known for their roles in several physiological and pathological processes. These factors are known to bind in vivo to BMP and TGF-ß receptors, respectively, which induces the phosphorylation of Smad (pSmad) transcription factors. This pathway is generally studied with Western blot and luciferase bioluminescence assay, which presents some limitations. PURPOSE: In this work, we developed and optimized a high-throughput assay to study pSmad pathways using immunofluorescence (IF) as an alternative to Western blot. We aimed to overcome the technical challenges usually faced in the classical IF assay in image acquisition, analysis, and quantification. METHODS: We used C2C12 cells as a cellular model. The cells were stimulated with BMP-2 and TGF-ß1 that were delivered either in solution (soluble) or via a biomaterial presenting the growth factor (GF), that is in a "matrix-bound" manner. Image acquisition parameters, analysis methods, and quantification of pSmads using IF were optimized for cells cultured on two types of supports: on bare glass and on a biomimetic coating made by self-assembly of the biopolymers hyaluronic acid and poly(l-lysine), which was crosslinked and then loaded with the GFs. RESULTS: We performed high-content kinetic studies of pSmad expression for cells cultured in 96-well microplates in response to soluble and matrix-bound BMP-2 and TGF-ß1. The detection limit of the IF-based assay was found to be similar to Western blot. Additionally, we provide a proof-of-concept for drug testing using inhibitors of BMP and TGF-ß receptors, under conditions where specific signaling pathways are engaged via the ligand/receptor interactions. Altogether, our findings offer perspectives for future mechanistic studies on cell signaling and for studies at the single cell level using imaging methods.

Confocal Microscopy of Fluorescently Labeled EHDV-Infected Cell Cultures.

The confocal laser scanning microscope allows the visualization of intracellular structures in greater detail than a widefield fluorescence microscope. Immunofluorescence (IF) techniques make use of the inherent ability of antibodies to bind to specific epitopes of specific proteins. Tagging these antibodies with an easily visualized molecule, e.g., a fluorophore, enables imaging in the fluorescence microscope. This is, however, a localization technique and will only give information about where certain proteins are; it does not provide the ultrastructural context provided by the transmission electron microscope. It also relies heavily on the accuracy and binding affinity of individual primary antibodies. Despite this, it is a commonly used, robust, and adaptable technique. In this chapter, we use a long-established IF protocol from our laboratory to locate EHDV proteins in a monolayer of infected cultured cells.

Methods for Monitoring ER-Phagy.

The endoplasmic reticulum (ER) serves as a central hub for protein synthesis, folding, and lipid biosynthesis in eukaryotic cells. Maintaining ER homeostasis is essential for optimal cellular function, and one mechanism that has garnered attention is endoplasmic reticulum-specific autophagy, or ER-phagy. ER-phagy selectively removes specific ER portions, playing a pivotal role in cellular health and adaptation to environmental stressors. ER-phagy can be induced by diverse cellular conditions such as amino acid starvation, disruption of ER quality control mechanisms, and accumulation of misfolded ER protein, highlighting cellular adaptability and the significance of ER-phagy in stress responses. Clinically relevant mutations in ER-phagy receptors are implicated in various diseases, underlining the fundamental importance of ER-phagy in ER homeostasis. Here, we provide comprehensive protocols and general considerations while investigating ER-phagy using three fundamental techniques-Western blotting, immunofluorescence, and flow cytometry-commonly used in ER-phagy detection and quantitation.

Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue.

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.

Analysis of toxicological findings related to immunofluorescence investigations.

Background: The subject of this article is the role of forensic toxicology in post-mortem examinations using immunofluorescence methods, its implications and its role in providing conclusive evidence for both criminal and civil proceedings. The aim of the study is to verify the correlation between the mode of death and the ingestion of exogenous substances and, if positive, to identify the category of substances ingested and assess their role in the cause of death. Materials and methods: A laboratory study was carried out, consisting of several phases: pre-analytical phase; analytical phase; post-analytical phase. The variables analyzed were sex, cause of death, age. Abused substances tested: amphetamines, methamphetamines, barbiturates, benzodiazepines, cocaine, methadone, opiates, tricyclic antidepressants, delta-9-tetrahydrocannabinol (cannabis), alcohol. Conclusions: Retrospective analysis was performed on a total sample of 55 cases. The most relevant data emerged: cocaine with an incidence of 7.3% (4 cases out of 55), amphetamines with 5.4% (3 cases in total). The results of the screening tests were then subjected to confirmatory tests. There is an association between the use of certain exogenous substances and an increased risk of certain causes of death, such as overdose, traffic accidents, cardiovascular deaths, etc. This paper has highlighted the possibility of using first level immunological tests, such as immunofluorescence, to provide preliminary answers to the judicial authority immediately after autopsy, and a quantitative deepening with further second level tests, such as gas chromatography, as a gold standard to determine the cause of death.

Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D.

Immunohistochemistry is a crucial method for detecting specific proteins within tissue samples, yet constrained to one biomarker per tissue section. Multiplexed immunofluorescence, while allowing simultaneous visualization of multiple proteins, faces limitations in the number of simultaneous fluorescent labels due to spectral overlap. Although cyclic immunofluorescence techniques have successfully broadened antibody staining capacities in a single tissue sample, they are plagued by time-consuming and labor-intensive procedures, sample degradation risks, and inability to scale beyond thin sections. In this study, we introduce the use of 3D confocal Fluorescence Lifetime Imaging Microscopy as a high-throughput, multiplexed immunofluorescence platform that can differentiate 11 or more biomarkers in 3D tissue volumes. Leveraging both spectral and lifetime information, this approach allows for practical spatial biology in thin sections that can readily scale to larger volumes of tissue. We believe that this highly multiplexed and versatile biomarker imaging platform will significantly expedite cancer research and enable new translational approaches in the future.

Features that characterize monoclonal light chain ("myeloma") cast nephropathy with immunofluorescence challenges and emphasis on electron microscopy.

Renal disease is a common cause of morbidity and mortality in patients with plasma cell dyscrasias. The serum-free light chain assay is used in patients, mostly older, with unexplained acute kidney injury to screen for potential myeloma cast nephropathy. This study consists of a systematic review of diagnostic features in myeloma cast nephropathy. The morphological features of tubular casts in patients with multiple myeloma have not been systematically analyzed. This study focuses on the morphology of these casts, emphasizing ultrastructural features, in a series of 23 patients with light chain ("myeloma") cast nephropathy and compared them with casts in 10 patients with various diseases. The immunofluorescence data were correlated with morphological findings to provide diagnostic assessments and practice guidelines. The ultrastructural features identified as diagnostic of casts associated with myeloma included: amyloid and crystals in the casts, multiple well-defined fracture planes forming a complex jigsaw puzzle arrangement of cast contents, indicative of the fragility of the immunoglobulin light chains involved, and reactive tubular cells lining the tubules with the casts. These features were seen in 95.2% of MCN cases and none of the casts in other renal conditions. Myeloma casts exhibited light chain monoclonality in a significant percentage of the MCN cases and often no staining for IgA or IgM. In contrast, the majority of non-myeloma casts stained for both kappa and lambda light chains, lgA, and lgM, and showed ultrastructurally a rather uniform finely to coarsely granular electron density occasionally admixed with cellular debris.

A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry.

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

A novel immunofluorescence study of Lingual Salivary Glands in the Egyptian Tortoise (Testudo kleinmanni) and its ecological significance.

The Egyptian tortoise (Testudo kleinmanni) is remarkably adapted to its harsh desert environment, a characteristic that is crucial for its survival under extreme conditions. This study was aimed at providing a deeper understanding of the lingual salivary gland structures in the Egyptian tortoise and examining how these structures help the tortoise manage hydration and nutrition in arid conditions. Utilizing a combination of light microscopy and immunofluorescence, this research introduced pioneering methods involving seven different antibodies, marking a first in the study of reptilian salivary glands. Our investigations categorized the tortoise's salivary glands into papillary and non-papillary types. The papillary glands were further classified into superficial, deep, interpapillary, and intraepithelial salivary glands, while non-papillary glands included superficial and deep lingual types. Structurally, these glands are organized into lobules, delineated by interlobular septa, and are equipped with a duct system comprising interlobular, intercalated, and main excretory ducts with gland openings on the tongue's surface and the papillae surfaces. Notably, the superficial glands displayed both tubuloalveolar and acinar configurations, whereas the deep lingual glands were exclusively acinar. Immunofluorescence results indicated that α-smooth muscle actin (α-SMA) was prevalent in myoepithelial cells, myofibroblasts, and blood vessels, suggesting their integral role in glandular function and support. E-cadherin was predominantly found in epithelial cells, enhancing cell adhesion and integrity, which are critical for efficient saliva secretion. Importantly, Mucin 1 (MUC1) and Mucin 5B (MUC5B) staining revealed that most glands were mucous in nature, with MUC5B specifically marking mucin within secretory cells, confirming their primary function in mucous secretion. PDGFRα and CD34 highlighted the presence of telocytes and stromal cells within the glandular and interlobular septa, indicating a role in structural organization and possibly in regenerative processes. Cytokeratin 14 expression was noted in the basal cells of the glands, underscoring its role in upholding the structural foundation of the epithelial barrier. In conclusion, this detailed morphological and immunological characterization of the Egyptian tortoise's salivary glands provides new insights into their complex structure and essential functions. These findings not only enhance our understanding of reptilian physiology but also underline the critical nature of salivary glands in supporting life in arid environments. This study's innovative use of a broad range of immunofluorescence markers opens new avenues for further research into the adaptive mechanisms of reptiles.

Virtual multiplexed immunofluorescence staining from non-antibody-stained fluorescence imaging for gastric cancer prognosis.

BACKGROUND: Multiplexed immunofluorescence (mIF) staining, such as CODEX and MIBI, holds significant clinical value for various fields, such as disease diagnosis, biological research, and drug development. However, these techniques are often hindered by high time and cost requirements. METHODS: Here we present a Multimodal-Attention-based virtual mIF Staining (MAS) system that utilises a deep learning model to extract potential antibody-related features from dual-modal non-antibody-stained fluorescence imaging, specifically autofluorescence (AF) and DAPI imaging. The MAS system simultaneously generates predictions of mIF with multiple survival-associated biomarkers in gastric cancer using self- and multi-attention learning mechanisms. FINDINGS: Experimental results with 180 pathological slides from 94 patients with gastric cancer demonstrate the efficiency and consistent performance of the MAS system in both cancer and noncancer gastric tissues. Furthermore, we showcase the prognostic accuracy of the virtual mIF images of seven gastric cancer related biomarkers, including CD3, CD20, FOXP3, PD1, CD8, CD163, and PD-L1, which is comparable to those obtained from the standard mIF staining. INTERPRETATION: The MAS system rapidly generates reliable multiplexed staining, greatly reducing the cost of mIF and improving clinical workflow. FUNDING: Stanford 2022 HAI Seed Grant; National Institutes of Health 1R01CA256890.

Changes of epidermal proteins immunolocalization in the corneous layer from embryonic to definitive avian beak.

The process of keratinization and cornification in the developing beak has been studied through immunofluorescence and immunogold electron microscopy in chick and zebrafinch embryos. After the curved beak anlagen appears at the tip of the maxillar bone, 5-8 layers of embryonic epidermis are generated from the basal layer of the epidermis. These cells are weakly immunoabeled for IFKs (Intermediate Filament Keratins) and more intensely for scaffoldin, a protein of the EDC (Epidermal Differentiation Complex) involved in the soft keratinization of the embryonic epidermis. Immunolabeling for CBPs (Corneous Beta Proteins) is visible in the transitional embryonic layers that are temporarily generated between the embryonic and definitive beak epidermis. The electron microscope reveals that intermediate layers contain immunolabeled periderm granules for scaffoldin mixed with bundles of corneous material immunolabeled for CBPs. Intense CBPs labeling occurs in the compacting corneous bundles of beta-keratinocytes in the definitive beak while scaffolding labeling disappears. The embryonic epidermis is sloughed before hatching. Sox (Sulfhydryl Oxidase) immunolabeling reveals that the enzyme is almost absent in embryonic layers but is present in transitional and definitive beta-keratinocytes. This indicates the formation of cross-linked disulfide bonds in the definitive corneous layer of the beak. Some calcium precipitation, suggested from von Kossa staining, occurs in the corneous layers only on the 18th day of development in the chick, in preparation for hatching.

Multiple approaches for the evaluation of connexin-43 expression and function in macrophages.

Connexins are essential gap junction proteins that play pivotal roles in intercellular communication in various organs of mammals. Connexin-43 (Cx43) is expressed in various components of the immune system, and there is extensive evidence of its participation in inflammation responses. The involvement of Cx43 in macrophage functionality involves the purinergic signaling pathway. Macrophages contribute to defenses against inflammatory reactions such as bacterial sepsis and peritonitis. Several assays can identify the presence and activity of Cx43 in macrophages. Real-time polymerase chain reaction (PCR) can measure the relative mRNA expression of Cx43, whereas western blotting can detect protein expression levels. Using immunofluorescence assays, it is possible to analyze the expression and observe the localization of Cx43 in cells or tissues. Moreover, connexin-mediated gap junction intercellular communication can be evaluated using functional assays such as microinjection of fluorescent dyes or scrape loading-dye transfer. The use of selective inhibitors contributes to this understanding and reinforces the role of connexins in various processes. Here, we discuss these methods to evaluate Cx43 and macrophage gap junctions.

Identification of circulating tumor cells captured by the FDA-cleared Parsortix® PC1 system from the peripheral blood of metastatic breast cancer patients using immunofluorescence and cytopathological evaluations.

Circulating Tumor Cells (CTCs) may serve as a non-invasive source of tumor material to investigate an individual's disease in real-time. The Parsortix® PC1 System, the first FDA-cleared medical device for the capture and harvest of CTCs from peripheral blood of metastatic breast cancer (MBC) patients for use in subsequent user-validated downstream analyses, enables the epitope-independent capture of CTCs with diverse phenotypes based on cell size and deformability. The aim of this study was to determine the proportion of MBC patients and self-declared female healthy volunteers (HVs) that had CTCs identified using immunofluorescence (IF) or Wright-Giemsa (WG) staining. Peripheral blood from 76 HVs and 76 MBC patients was processed on Parsortix® PC1 Systems. Harvested cells were cytospun onto a charged slide and immunofluorescently stained for identification of CTCs expressing epithelial markers. The IF slides were subsequently WG-stained and analyzed for CTC identification based on morphological features of malignant cells. All testing was performed by operators blinded to the clinical status of each subject. CTCs were identified on the IF slides in 45.3% (≥ 1) / 24.0% (≥ 5) of the MBC patients (range = 0 - 125, mean = 7) and in 6.9% (≥ 1) / 2.8% (≥ 5) of the HVs (range = 0 - 28, mean = 1). Among the MBC patients with ≥ 1 CTC, 70.6% had only CK + /EpCAM- CTCs, with none having EpCAM + /CK- CTCs. CTC clusters were identified in 56.0% of the CTC-positive patients. On the WG-stained slides, CTCs were identified in 42.9% (≥ 1) / 21.4% (≥ 5) of the MBC patients (range = 0 - 41, mean = 4) and 4.3% (≥ 1) / 2.9% (≥ 5) of the HVs (range = 0 - 14, mean = 0). This study demonstrated the ability of the Parsortix® PC1 System to capture and harvest CTCs from a significantly larger proportion of MBC patients compared to HVs when coupled with both IF and WG cytomorphological assessment. The presence of epithelial cells in subjects without diagnosed disease has been previously described, with their significance being unclear. Interestingly, a high proportion of the identified CTCs did not express EpCAM, highlighting the limitations of using EpCAM-based approaches.

Performance of a point-of-care test for the detection of anti-Leishmania infantum antibodies is associated with immunofluorescent antibody titer and clinical stage of leishmaniosis in dogs from endemic regions.

Canine leishmaniosis (CanL) is caused by the protozoal parasite Leishmania infantum, which is transmitted by sand flies in warm climates across the world. Because dogs are considered a primary domestic reservoir for the parasite that causes leishmaniosis in humans, it is important from a One Health perspective that CanL be properly managed. In endemic regions, CanL is a common differential diagnosis in sick dogs because the clinical signs and clinicopathological disorders of the disease are non-specific, variable, and may overlap those of other common conditions. Diagnosis is based on the presence of compatible clinical signs, laboratory abnormalities, and confirmation by serological and parasitological evidence of infection. Here, we describe the performance of a point-of-care (POC) immunoassay that uses recombinant antigens to detect canine anti- L. infantum antibodies in a convenience sample set from a diagnostic laboratory, a group of canine patients with clinical staging, and in apparently healthy dogs from endemic areas. An immunofluorescence antibody test (IFAT) was used as the semiquantitative reference method. In the convenience sample set with high IFAT titers (≥ 1:800), the POC immunoassay demonstrated perfect agreement with IFAT (100%; 90/90). Using samples from dogs staged as either LeishVet Stage 2 or 3 or LeishVet Stage 1, positive agreement of the POC immunoassay with the IFAT was 98.8% (82/83) and 83.8% (31/37), respectively. The negative agreement with IFAT was 98.9% (272/275) in apparently healthy dogs from endemic areas of Greece and Italy. Since the performance of the POC immunoassay was associated with IFAT titer and clinical stage of CanL, the test may help veterinarians when determining if CanL is likely responsible for a patient's clinical picture or when evaluating an apparently healthy patient prior to vaccination.

Optimizing immunofluorescence with high-dynamic-range imaging to enhance PD-L1 expression evaluation for 3D pathology assessment from NSCLC tumor tissue.

Assessing programmed death ligand 1 (PD-L1) expression through immunohistochemistry (IHC) is the golden standard in predicting immunotherapy response of non-small cell lung cancer (NSCLC). However, observation of heterogeneous PD-L1 distribution in tumor space is a challenge using IHC only. Meanwhile, immunofluorescence (IF) could support both planar and three-dimensional (3D) histological analyses by combining tissue optical clearing with confocal microscopy. We optimized clinical tissue preparation for the IF assay focusing on staining, imaging, and post-processing to achieve quality identical to traditional IHC assay. To overcome limited dynamic range of the fluorescence microscope's detection system, we incorporated a high dynamic range (HDR) algorithm to restore the post imaging IF expression pattern and further 3D IF images. Following HDR processing, a noticeable improvement in the accuracy of diagnosis (85.7%) was achieved using IF images by pathologists. Moreover, 3D IF images revealed a 25% change in tumor proportion score for PD-L1 expression at various depths within tumors. We have established an optimal and reproducible process for PD-L1 IF images in NSCLC, yielding high quality data comparable to traditional IHC assays. The ability to discern accurate spatial PD-L1 distribution through 3D pathology analysis could provide more precise evaluation and prediction for immunotherapy targeting advanced NSCLC.

Immunodetection of tubulin and centromeric histone H3 (CENH3) proteins in Glycine species.

BACKGROUND: The centromeres appear as primary constrictions on monocentric metaphase chromosomes; where sister chromatids are held together and assemble the proteinaceous kitechore complex at which microtubule proteins attach during nuclear divisions for pulling sister chromatids to opposite cell poles. The movement of chromosomes is usually governed by structural proteins that are either species-specific or highly conserved, such as the centromere-specific histone H3 (CENH3) and tubulin proteins, respectively. METHODS AND RESULTS: We aimed to detect these proteins across eight different Glycine species by an immunofluorescence assay using specific antibodies. Furthermore, with the α-tubulin antibody we traced the dynamics of microtubules during the mitotic cell cycle in Glycine max. With two-color immunofluorescence staining, we showed that both proteins interact during nuclear division. CONCLUSIONS: Finally, we proved that in different diploid and tetraploid Glycine species CENH3 can be detected in functional centromeres with spatial proximity of microtubule proteins.