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Aspirin is one of the most frequently detected non-steroidal anti-inflammatory drugs in aquatic environments. Despite its prevalence, toxicity possessed by aspirin to non-target organisms like fish is poorly explored. In the present study, cell death induced by different concentrations of aspirin (1, 10, and 100 µg/L) has been investigated in the liver of fish, Labeo rohita exposed for 28 days. A significant increase (p < 0.05) in the density of caspase-3 positive cells in a dose and duration-dependent manner assessed through immunofluorescent staining indicates caspase-dependent pathway of cell death which may be either through intrinsic or extrinsic pathway. The flow cytometric analysis, in addition, revealed a significant (p < 0.05) decline in the live cells and an increase in apoptotic cells in the liver of fish exposed to aspirin. Cell death due to apoptosis is further indicated by a significant increase (p < 0.05) in the Kupffer cells and tumor necrosis factor-α. The decrease in the activity of cyclooxygenase (COX) enzyme significantly (p < 0.05) in all three exposure concentrations of aspirin suggests COX-dependent pathway of cell death. The present study provides in-depth insights into aspirin-induced cell death in the liver of fish at environmentally realistic concentrations.

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ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADPRylation. Recently we developed a set of recombinant antibody-like ADP-ribose (ADPR) binding proteins using naturally occurring ADPR binding domains (ARBDs), including macrodomains and WWE domains, functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with the sequence from two other species, mouse and goat. These new reagents are based on a previously characterized set of naturally occurring ARBDs with known specificity. Characterization of the new reagents demonstrates that they can be detected in a species-dependent manner by secondary immunological tools, recognize specific ADPR moieties, and can be used for simultaneous detection of mono ADPR and poly ADPR at single-cell resolution in various antibody-based assays. The expansion of this toolkit will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.

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Here, we describe immunofluorescent (IF) staining assay of 3D cell culture colonoids isolated from mice colon as described previously. Primary cultures developed from isolated colonic stem cells are called colonoids. Immunofluorescence can be used to analyze the distribution of proteins, glycans, and small molecules-both biological and non-biological ones. Four-day-old colonoid cell cultures grown on Lab-Tek 8-well plate are fixed by paraformaldehyde. Fixed colonoids are then subjected to antigen retrieval and blocking followed by incubation with primary antibody. A corresponding secondary antibody tagged with desired fluorescence is used to visualize primary antibody-marked protein. Counter staining to stain actin filaments and nucleus to assess cell structure and DNA in nucleus is performed by choosing the other two contrasting fluorescences. IF staining of colonoids can be utilized to visualize molecular markers of cell behavior. This technique can be used for translation research by isolating colonoids from colitis patients' colons, monitoring the biomarkers, and customizing their treatments. Key features • Analysis of molecular markers of cell behavior. • Protocol to visualize proteins in 3D cell culture. • This protocol requires colonoids isolated from mice colon grown on matrigel support. • Protocol requires at least eight days to complete.

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The mammalian integumentary system, including skin and its appendages, serves as a protective barrier for the body. During development, skin epidermis undergoes rapid cell division and differentiation to form multiple stratified layers of keratinocytes. Concurrently the epidermis also gives rise to hair follicles that invaginate into the dermis. In adult skin, the hair follicle undergoes cyclic regeneration fueled by hair follicle stem cells located in the bulge. Three-dimensional and high-resolution imaging of these structures using whole-mount immunofluorescent staining allows to better characterize epidermal progenitors and stem cells.

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INTRODUCTION: Inward rectifier K+ (Kir) channel, a major factor determining endothelial membrane potential, regulates Ca2+ influx and vasodilator release, which is impaired in preeclamptic blood vessels. Previously, human umbilical vein endothelial cell (HUVEC) Kir currents were shown to decrease after incubating in preeclamptic plasma. We aimed to demonstrate whether sFlt-1, which is high in preeclamptic blood, could inhibit Kir channel function and expression. METHODS: HUVECs were cultured in regular medium, regular medium with added sFlt-1, or serum from preeclampsia patients or normal pregnant women (Control, sFlt-1, PE, or NP, respectively). Using whole-cell patch clamp technique, we identified Kir currents with the Kir blocker 2 mM BaCl2 and compared the currents among groups. The expression of Kir 2.1 and 2.2 channels were determined using immunofluorescent staining. RESULTS: sFlt-1 and PE groups exhibited similar Kir currents, while NP group possessed significantly larger currents, similar to Control group currents. Moreover, sFlt-1 and sFlt-1/PlGF ratio showed strong negative correlation with Kir currents (r = -0.71 and -0.70, respectively; P < 0.05). There were no significant differences in mean fluorescence intensity representing Kir 2.1 and 2.2 channels expression in all four groups. DISCUSSION: This is the first report to demonstrate sFlt-1 inhibition against Kir currents, which could lead to maternal endothelial dysfunction and hypertension seen in preeclampsia. However, channel expression was unaffected by sFlt-1 incubation, suggesting dysfunctions of channel or other processes (e.g., membrane translocation). The present data could pave the way for novel therapies targeting sFlt-1 or Kir to alleviate hypertension in preeclampsia.

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In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols.

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The use of polychromatic immunofluorescent staining on whole-mount skin enables cell type characterization and aids in the delineation of the physiological and immunological strategies used by the skin to combat pathogens. Using whole-mount skin for polychromatic immunofluorescent staining removes the need for histological sectioning and enables the visualization of anatomical structures and immune cell types in three dimensions. Here we present a detailed protocol for immunostaining with fluorescence-conjugated primary antibodies in whole-mount skin to reveal structural landmarks and specific immune cell types using confocal laser scanning microscopy (CLSM) (Basic Protocol 1). The optimized staining panel reveals structural features such as blood vessels (CD31 antibody) and the lymphatic network (LYVE-1 antibody), in combination with MHCII antibodies for antigen-presenting cells (APCs), CD64 for macrophages and monocytes, CD103 for dendritic epidermal T cells (DETC), and CD326 for Langerhans cells (LC). Basic Protocol 2 describes image visualization pipelines using open-source software (ImageJ/FIJI), enabling four visualization options (z-projections, orthogonal views, 3D visualization, and animation). Basic Protocol 3 describes a quantitative analysis pipeline using CellProfiler to characterize the spatial relationship between cell types using mathematical indices such as Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME). These protocols will enable researchers to stain, record, analyze, and interpret data from whole-mount skin using commercially available reagents in a CLSM-equipped laboratory and freely available analysis software. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescent staining and imaging for whole-mount mouse skin Basic Protocol 2: File rendering and visualization using FIJI Basic Protocol 3: Spatial image analysis using CellProfiler.

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BACKGROUND: To investigate the expression of elastin in the conjoint facial sheath (CFS) in patients with severe unilateral congenital blepharoptosis in different age groups. METHODS: Twenty-seven cases of severe unilateral congenital blepharoptosis (27 eyes) were treated with CFS + LM complex suspension from January 2020 to July 2020. Within that sample, 9 patients were over 18 years old, 9 patients were 13 to 17 years old and 9 patients were 5 to 12 years old. CFS and LM specimens were collected during CFS + LM complex suspension surgery. In the CFS specimens, the elastic fibers were observed by Victoria Blue staining. The elastin expression levels of the three groups of specimens were determined and analyzed by immunofluorescent staining and Western blotting. RESULTS: Victoria Blue staining showed that elastic fibers were abundant in CFS tissue. Moreover, immunofluorescent staining showed strong positive expression of elastin in the CFS and LM. Furthermore, in the child group, the Western blot results demonstrated that the expression of elastin was higher in the CFS than in the LM (P < 0.05). Additionally, the expression of elastin was significantly higher in the CFS of children than in that of adults or adolescents (P < 0.001). CONCLUSIONS: The CFS and LM are rich in elastic fibers and elastin, although elastin expression in the CFS decreases with age. Thus, it is feasible to apply CFS + LM complex suspension to cure severe unilateral congenital blepharoptosis.

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We described Pelomyxa doughnuta sp. nov. and examined it with the use of light, electron, and immunofluorescence microscopy as well as cytochemical methods. The cells of P. doughnuta sp. nov. are usually binuclear, although cells with one, three, or four nuclei are sometimes found in the population. A unique feature of the new species is a dense capsule around the nucleus. It consists of a continuous layer of glycogen 5-20 µm thick. The tubulin cytoskeleton is mainly represented by perinuclear microtubules. P. doughnuta sp. nov. has a filamentous glycocalyx and strongly reduced components of flagellar apparatus. Obligate prokaryotic endocytobionts of two morphotypes are present in the cytoplasm.

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Infectious laryngotracheitis virus (ILTV) DNA has been detected in blood fractions, but the cell phenotype with which the virus is associated is unknown. This study investigated the presence of ILTV antigen in peripheral blood cells of six acutely ILTV-infected chickens (5 or 9 days post ocular inoculation with a virulent isolate) and three sham-inoculated chickens using immunofluorescent staining. Blood fractions were separated by Ficoll-Paque density gradient centrifugation, and smears were prepared from erythrocyte and leukocyte fractions. The smears were stained for ILTV glycoprotein E and the leukocyte markers CD4, CD8, Bu-1 (B cell), KUL01 (monocyte/macrophage), TCRγδ, and TCRαß/Vß2 and examined under a confocal microscope. In samples from infected birds, ILTV gE-specific fluorescence was localized in B cells and all evaluated T cell types, but not in monocytes and erythrocytes. The percentage of CD4, CD8, TCRγδ, TCRαß/Vß1, TCRαß/Vß2 and B cells positive for ILTV antigen ranged from 13.3% to 22.3%. None of the samples from the sham-inoculated chickens exhibited fluorescence for ILTV gE. The results of this pilot study suggest that ILTV has a tropism for peripheral blood T and B cells. Further research is required to investigate whether these cells support ILTV productive replication. RESEARCH HIGHLIGHTSSelective tropism of ILTV for peripheral blood cells was demonstrated in acutely infected birds.The ILTV antigen gE was detected in blood CD4, CD8, TCRγδ, TCRαß and B cells but not in monocytes and erythrocytes.The highest percentage of ILTV antigen was observed in CD4 cells (22.3%) followed by TCRαß/Vß1 (20.6%), CD8 (15.4%), TCRαß/Vß2 or B cells (14.4%) and TCRγδ cells (13.3%).

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BACKGROUND AND AIM: Local breeds of chicken are known to have relatively higher disease resistance to many endemic diseases and diseases that are highly virulent in commercial chickens. This study aimed to address the lymphocyte subpopulations in three constitutive immune system organs (thymus, bursa of Fabricius, and spleen) in 30, 8-week-old, male local breed chickens. MATERIALS AND METHODS: The T (CD3+) and B lymphocytes (Bu-1+) were identified through one-color, direct immunofluorescent staining of the thymus, bursa, and spleen lymphocytes. Likewise, two-color, direct immunofluorescent staining was performed to identify the CD4- and/or CD8-defined T lymphocytes. The proportions of T and B lymphocytes and CD4- and/or CD8 defined chicken lymphocyte subsets in lymphoid suspensions prepared from the thymus, bursa, and spleen were determined by flow cytometry. RESULTS: CD3+ cells, particularly those positive for CD4+CD8-, were dominant in the thymus, whereas cells expressing the Bu-1 marker were predominant in the bursa of Fabricius. The proportion of T and B cells was almost equal in the spleen, with more cells expressing the CD4-CD8+ marker in the red pulp. CONCLUSION: These findings indicate that local breeds of chicken could serve as a reliable model for studying the immune system of commercial light chicken breeds, due to the similarity in the presence and the distribution of the immune cells.

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Parkinson's disease (PD) is a neurodegenerative disorder that affects the motor system. PD is characterized by the accumulation of intracellular protein aggregates, Lewy bodies, and Lewy neurites, composed primarily of the protein α-synuclein. Thus, PD is classified as the most common synucleinopathy. The motor symptoms of the disease result from the death of cells in the region of the midbrain, leading to a dopamine deficit. While the cause of PD is unknown, it is believed to involve both inherited and environmental factors. PD has been extensively studied using in vitro and in vivo models; however, some discrepancy is observed in these results. In order to analyze progressive neurodegenerative disease, experimental platform amenable to continuous observation and experimental manipulation is required. In this chapter, we provide a practical method to slice and cultivate the midbrain tissue as an ex vivo experimental model.

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This study explored whether KMUP-1 improved chronic constriction injury (CCI)-induced BKCa current inhibition in dorsal root ganglion (DRG) neurons. Rats were randomly assigned to four groups: sham, sham + KMUP-1, CCI, and CCI + KMUP-1 (5 mg/kg/day, i.p.). DRG neuronal cells (L4-L6) were isolated on day 7 after CCI surgery. Perforated patch-clamp and inside-out recordings were used to monitor BKCa currents and channel activities, respectively, in the DRG neurons. Additionally, DRG neurons were immunostained with anti-NeuN, anti-NF200 and anti-BKCa. Real-time PCR was used to measure BKCa mRNA levels. In perforated patch-clamp recordings, CCI-mediated nerve injury inhibited BKCa currents in DRG neurons compared with the sham group, whereas KMUP-1 prevented this effect. CCI also decreased BKCa channel activity, which was recovered by KMUP-1 administration. Immunofluorescent staining further demonstrated that CCI reduced BKCa-channel proteins, and KMUP-1 reversed this. KMUP-1 also changed CCI-reduced BKCa mRNA levels. KMUP-1 prevented CCI-induced neuropathic pain and BKCa current inhibition in a peripheral nerve injury model, suggesting that KMUP-1 could be a potential agent for controlling neuropathic pain.

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Helicobacter pylori chronically infects the gastric mucosa of humans and diseases associated with infection include gastritis, peptic ulceration, and development of gastric cancer. The organism displays a distinct tropism for the gastric mucosa of humans and for the gastric mucin MUC5AC. While the majority of organisms are found in the mucus layer overlying the epithelial cells in the stomach, adherence of the organism to the gastric epithelium is necessary for the development of disease. The interaction of H. pylori with epithelial cells results in subversion of host cell signaling and induction of an inflammatory response. Factors that influence the outcome of infection include host genetics, environmental factors, and the phenotype of the infecting strain. In this chapter, we describe cell culture assays to assess the interaction of H. pylori with epithelial cells, immunofluorescent staining to detect H. pylori in infected human gastric biopsy specimens and the use of flow cytometry to detect mucin binding to H. pylori.

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The biophysical properties of cells change with cancer invasion to fulfill their metastatic behavior. Cell softening induced by cancer is highly associated with alterations in cytoskeleton fibers. Changes in the mechanical properties of cytoskeletal fibers have not been quantified due to technical limitations. In this study, we used the micropipette aspiration technique to calculate and compare the viscoelastic properties of non-invasive and invasive breast cancer cells. We evaluated the mechanical properties of actin fibers and microtubules of two cancerous cell lines by using multiscale tensegrity modeling and an optimization method. Cancer invasion caused altered viscoelastic behavior of cells and the results of modeling showed changes in mechanical properties of major cytoskeleton fibers. The stiffness and viscosity constant of actin fibers in non-invasive cells were 1.28 and 2.27 times higher than those of the invasive cells, respectively. However, changes in mechanical properties of microtubules were minor. Immunofluorescent staining of fibers and their quantified distributions confirmed altered actin distribution among two cell lines, in contrast to microtubule distribution. This study highlights the function of cytoskeletal fibers in cancer progression, which could be of interest in designing therapeutic strategies to target cancer progress. Firstly, the viscoelastic behavior of non-invasive and invasive cells is examined with micropipette aspiration tests. A tensegrity model of cells is developed to mimic the viscoelastic behavior of cells, and tensegrity element stiffness is evaluated in an optimization procedure based on micropipette aspiration tests. Finally, by using immunofluorescent staining and confocal imaging, mechanical properties of actin filaments and microtubules of cancer cells are investigated during the course of metastasis.

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Circulating tumor cells (CTCs) indicate the diagnosis and prognosis of cancer patients, together with benefiting individual treatment and anticancer drug development. However, their large-scale application in general population still requires systematically multifaceted modifications for currently proprietary new technologies based on filtration. We primitively utilized a cell size-based platform to evaluate the recovery efficiency of spiked abnormal cell lines and analyzed circulating abnormal cells (CACs). To dissect the subpopulations of CACs, we conducted immunofluorescent (IF) staining with a combination of unique biomarkers of CTCs and circulating endothelial cells (CECs). Furthermore, we improved the CTC screening system by assessing the feasibility of transferring CTCs for automatic IF analysis, together with simulating and optimizing the circumstances for long-term CTC storage and transportation. We detected CACs in 15 HD candidates with CTC characteristics such as abnormally large cytomorphology, high nuclear-cytoplasmic ratio, and positive for panCK or VIM staining. Thereafter, we improved accuracy of the platform by distinguishing CTCs from CECs, which satisfied the elementary requirement for small-scale CTC screening in HD candidates. Finally, large-scale CTC screening in general population was available after multifaceted modifications including automatic analysis by transferring CTCs on slides, choosing the appropriate blood-collecting tube, optimizing the conditions for long-term CTC storage and transportation, and evaluating the potential effect on the CTC phenotype. Hence, we systematically modified the scope of technique parameters, improved the accuracy of early cancer detection, and made it realizable for large-scale CTC or CEC screening in general population.

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Approximately 5% of patients with IgA nephropathy (IgAN) exhibit mild mesangial lesions with acute onset nephrotic syndrome and diffuse foot process effacement representative of minimal change disease (MCD). It is not clear whether these unusual cases of IgAN with MCD (IgAN-MCD) are variant types of IgAN or coincidental deposition of IgA in patients with MCD. In a retrospective multicenter cohort study of 18 hospitals in Korea, we analyzed 46 patients with IgAN-MCD. Patients with endocapillary proliferation, segmental sclerosis, and crescent were excluded, and the clinical features and prognosis of IgAN-MCD were compared with those of pure MCD. In addition, we performed galactose-deficient IgA1 (KM55) staining to characterize IgAN-MCD. Among the 21,697 patients with glomerulonephritis enrolled in the database, 46 patients (0.21%) were diagnosed with IgAN-MCD, and 1610 patients (7.4%) with pure MCD. The 46 patients with IgAN-MCD accounted for 0.6% of primary IgAN patients (n = 7584). There was no difference in prognosis between patients with IgAN-MCD and those with only MCD. IgA and KM55 showed double positivity in all patients with IgAN-MCD (n = 4) or primary IgAN (n = 5) under double immunofluorescent staining. However, in four patients with lupus nephritis, mesangial IgA was deposited, but galactose-deficient-IgA1 (Gd-IgA1) was not. These findings suggest that IgAN-MCD is a dual glomerulopathy in which MCD was superimposed on possibly indolent IgAN. We confirmed by KM55 staining that IgAN-MCD is true IgAN, enabling better characterizations of the disease. Furthermore, IgAN-MCD shows a good prognosis when treated according to the usual MCD treatment modality.

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Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows the assessment of changes in their localization and abundance. We use the described protocol to stain claudin-2, but it can also be adapted to stain any tight junction protein. We found that using methanol for fixing allows the best preservation of claudin-2 both at the membrane and in cytoplasmic vesicles. Staining is done using a claudin-2 specific primary and a fluorescently labelled secondary antibody, along with DAPI to label nuclei. The samples are then imaged using confocal microscopy, and a z-stack is obtained allowing visualization of both junctional and intracellular claudin-2. Total claudin-2 signal can be quantified after 3D reconstruction of the images using the Imaris software.

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Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.

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Amphetamine (AMPH), an appetite suppressant, alters expression levels of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. This study explored the potential role of cJun-N-terminal kinases (JNK) in appetite control, mediated by reactive oxygen species (ROS) and activator protein-1 (AP-1) in AMPH-treated rats. Rats were given AMPH daily for 4 days. Changes in feeding behavior and expression levels of hypothalamic NPY, CART, cFos, cJun, phosphorylated JNK (pJNK), as well as those of anti-oxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione S-transferase (GST), were examined and compared. Following AMPH treatment, food intake and NPY expression decreased, whereas the other proteins expression and AP-1/DNA binding activity increased. Both cerebral cJun inhibition and ROS inhibition attenuated AMPH anorexia and modified detected protein, revealing a crucial role for AP-1 and ROS in regulating AMPH-induced appetite control. Moreover, both pJNK/CART and SOD/CART activities detected by double immunofluorescent staining increased in hypothalamic arcuate nucleus in AMPH-treated rats. The results suggested that pJNK/AP-1 signaling and endogenous anti-oxidants participated in regulating NPY/CART-mediated appetite control in rats treated with AMPH. These findings advance understanding of the molecular mechanism underlying the role of pJNK/AP-1 and oxidative stress in NPY/CART-mediated appetite suppression in AMPH-treated rats.

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