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.
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.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS.
OBJECTIVE: Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent.
METHODS: Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA.
CONCLUSIONS: For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays.
CONCLUSIONS: The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.
Введение. Геморрагическая лихорадка с почечным синдромом (ГЛПС) является наиболее распространенным зоонозным вирусным заболеванием человека на территории Российской Федерации. Более 98% заболеваемости ГЛПС вызвано ортохантавирусом Пуумала (ПУУ). Для лабораторной диагностики ГЛПС, в частности серодиагностики клинических случаев, требуются эффективные (высокочувствительные, специфичные, максимально объективные и быстрые в исполнении) серологические тесты, разработка которых является важнейшим элементом контроля данного вирусного заболевания. Цель исследования. Конструирование системы иммуноферментного анализа (ИФА) для определения специфических антител с использованием стандартного антигена в виде высокоочищенного инактивированного вируса ПУУ в качестве иммуносорбента. Материалы и методы. Получение препаратов высокоочищенного антигена вируса ПУУ, конструирование системы ИФА для определения специфических антител, отработка параметров системы ИФА, параллельное титрование сывороток крови больных ГЛПС методом флуоресцирующих антител (МФА) и новым вариантом ИФА. Результаты и обсуждение. Впервые в лабораторной практике исследования ГЛПС была сконструирована система ИФА на основе очищенного инактивированного вируса ПУУ (целевой компонент экспериментальной вакцины против ГЛПС) в качестве стандартного антигена при прямой сорбции на твердую фазу (иммунопанель). Параллельное титрование 50 образцов сывороток крови больных ГЛПС методами МФА и разработанного варианта ИФА показало высокую чувствительность и специфичность данного варианта ИФА, отмечены 100% совпадение результатов тестов (на уровне положительный/отрицательный результат) и значительный уровень корреляции величин титров специфических антител двух тестов. Заключение. Разработанный вариант ИФА для определения антител к вирусу ПУУ на основе очищенного инактивированного вируса ПУУ – целевого компонента вакцинного препарата против ГЛПС в качестве иммуносорбента – может быть эффективно использован для серодиагностики ГЛПС и массовых серо-эпидемиологических исследований.

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.

Objective To explore a simple and feasible method for whole-mount immunofluorescence staining of lymphatic vessels in the ApoE-/- mouse model of atherosclerosis. Methods Aortic specimens were carefully excised from the ApoE-/- mouse model. Following immunostaining with specific antibodies against smooth muscle actin (SMA) and lymphatic vessel endothelial receptor 1 (LYVE1), the aortas, including the aortic root, were subjected to a 30-minute treatment with 5 g/L Sudan Black B solution. This step was instrumental in minimizing the autofluorescent background of the tissue. Thereafter, the aortas were processed through a clearing protocol and imaged within a purpose-built chamber under a fluorescence microscope. Results The pretreatment with 5 g/L Sudan Black B effectively suppressed the autofluorescent signals emanating from the vascular structures, thereby enhancing the contrast and clarity of the specific fluorescence signals associated with the lymphatic vessels. This enhancement in signal quality did not compromise the integrity or specificity of the immunofluorescent markers. Conclusion A facile, highly specific, and effective approach for the visualization of lymphatic vessels in whole-mount aortic preparations from ApoE-/- mice is established.

Tyrosine-protein kinase SYK, encoded by the SYK gene, is a non-receptor type protein kinase which mediates immune signal transduction through immunoreceptors. Tyrosine-protein kinase SYK expression has been associated with the development of various inflammatory diseases, cancer and neurodegenerative conditions. The reproducibility of tyrosine-protein kinase SYK research would help elucidate the mechanism in which it causes neuroinflammation as well as its potential as a novel target to treat Alzheimer\'s disease. This would be facilitated with the availability of high-quality tyrosine-protein kinase SYK. In this study, we characterized thirteen tyrosine-protein kinase SYK 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. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Breast cancer poses a global health challenge, yet the influence of ethnicity on the tumor microenvironment (TME) remains understudied. In this investigation, we examined immune cell infiltration in 230 breast cancer samples, emphasizing diverse ethnic populations. Leveraging tissue microarrays (TMAs) and core samples, we applied multiplex immunofluorescence (mIF) to dissect immune cell subtypes across TME regions. Our analysis revealed distinct immune cell distribution patterns, particularly enriched in aggressive molecular subtypes triple-negative and HER2-positive tumors. We observed significant correlations between immune cell abundance and key clinicopathological parameters, including tumor size, lymph node involvement, and patient overall survival. Notably, immune cell location within different TME regions showed varying correlations with clinicopathologic parameters. Additionally, ethnicities exhibited diverse distributions of cells, with certain ethnicities showing higher abundance compared to others. In TMA samples, patients of Chinese and Caribbean origin displayed significantly lower numbers of B cells, TAMs, and FOXP3-positive cells. These findings highlight the intricate interplay between immune cells and breast cancer progression, with implications for personalized treatment strategies. Moving forward, integrating advanced imaging techniques, and exploring immune cell heterogeneity in diverse ethnic cohorts can uncover novel immune signatures and guide tailored immunotherapeutic interventions, ultimately improving breast cancer management.

Immunofluorescence with antibodies against phosphorylated forms of H2AX (γH2AX) is revolutionizing our understanding of repair and signaling of DNA double-strand breaks (DSBs). Unfortunately, the pattern of γH2AX foci depends upon a number of parameters (nature of stress, number of foci, radiation dose, repair time, cell cycle phase, gene mutations, etc…) whose one of the common points is chromatin condensation/decondensation. Here, we endeavored to demonstrate how chromatin conformation affects γH2AX foci pattern and influences immunofluorescence signal. DSBs induced in non-transformed human fibroblasts were analyzed by γH2AX immunofluorescence with sodium butyrate treatment of chromatin applied after the irradiation that decondenses chromatin but does not induce DNA breaks. Our data showed that the pattern of γH2AX foci may drastically change with the experimental protocols in terms of size and brightness. Notably, some γH2AX minifoci resulting from the dispersion of the main signal due to chromatin decondensation may bias the quantification of the number of DSBs. We proposed a model called \"Christmas light models\" to tentatively explain this diversity of γH2AX foci pattern that may also be considered for any DNA damage marker that relocalizes as nuclear foci.

Spatial transcriptomics measures in situ gene expression at millions of locations within a tissue1, hitherto with some trade-off between transcriptome depth, spatial resolution and sample size2. Although integration of image-based segmentation has enabled impactful work in this context, it is limited by imaging quality and tissue heterogeneity. By contrast, recent array-based technologies offer the ability to measure the entire transcriptome at subcellular resolution across large samples3-6. Presently, there exist no approaches for cell type identification that directly leverage this information to annotate individual cells. Here we propose a multiscale approach to automatically classify cell types at this subcellular level, using both transcriptomic information and spatial context. We showcase this on both targeted and whole-transcriptome spatial platforms, improving cell classification and morphology for human kidney tissue and pinpointing individual sparsely distributed renal mouse immune cells without reliance on image data. By integrating these predictions into a topological pipeline based on multiparameter persistent homology7-9, we identify cell spatial relationships characteristic of a mouse model of lupus nephritis, which we validate experimentally by immunofluorescence. The proposed framework readily generalizes to new platforms, providing a comprehensive pipeline bridging different levels of biological organization from genes through to tissues.

The mandatory usage of extracellular matrix (ECM) gels in 3D cultures limits antibody penetration and increases background, while the removal of ECM gel causes disruption of morphology and sample loss. These factors pose challenges to effective immune labeling-based staining. Here, we present a protocol for whole-mount immunofluorescence staining of gel-embedded pancreatic organoids. We describe steps for sample fixation, blocking, and antibody incubation. We detail procedures for washing antibodies and mounting.

CONCLUSIONS: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.