3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization.

RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.

Approach to Kidney Biopsy: Core Curriculum 2022.

The kidney biopsy is an essential tool for diagnosis of many kidney diseases. Obtaining an adequate biopsy sample with appropriate allocation for various studies is essential. Nephrologists should understand key lesions and their interpretation because these are essential elements underlying optimal approaches for interventions. This installment in the AJKD Core Curriculum in Nephrology will review these topics. We will first briefly discuss considerations for allocation and processing of kidney biopsies. We will then present in outline form the differential diagnoses of a spectrum of patterns of injury and consideration for interpretation of specific lesions. Lesions are presented according to anatomic site as glomerular, vascular, or tubulointerstitial. Native and transplant kidney biopsy lesions are included. These lesions and differential diagnoses and specific diseases are then linked to detailed clinicopathologic discussion of specific diseases presented in the AJKD Atlas of Kidney Pathology II. Correlation with immunofluorescence, electron microscopy, and clinical findings are emphasized to reach a differential diagnosis and the final diagnosis.

Immunophenotyping: Application to Safety Assessment.

A continuing education course entitled "What You Always Wanted to Know About Immunotoxicology in Pharmaceutical Development…But Were Afraid to Ask" was offered at the Society of Toxicologic Pathology (STP) 36th annual symposium in Montreal. This article summarizes some key points made during the presentation dedicated to immunophenotyping. It describes how clusters of differentiation (CDs) are well-defined antigens used to characterize cell subsets, and how lymphocyte subsets in humans and different rodent and nonrodent species can be defined by detection of various combinations of CDs. It provides an overview of immunophenotyping study design considerations and applications to safety assessment.

Generation and application of a novel InsP(3)R(1) mono-antibody from mouse.

Inositol 1, 4, 5-Trisphosphate Receptor (InsP3R) is an intracellular Ca(2+) release channel, which widely participates in cellular processes. Three isoforms of InsP3R were identified as InsP3R1, InsP3R2, and InsP3R3. They share 60-0% protein sequence homology and form a channel in a manner of homotetramer or heterotetramer. Several InsP3R isoform-specific rabbit antibodies have been generated to distinguish their localization and functions. However, there is no report of such a valid antibody raised from other species. In his article, we prepare a mouse monoclonal antibody against a synthetic peptide with rat InsP3R1-specific carboxyl terminus sequence. This monoclonal antibody of InsP3R1 (R1-mAb) was purified and characterized as IgG2b, which can recognize InsP3R1 by Western-blot (WB) analysis and immunoprecipitate (IP) InsP3R1 from moue brain lysate tested. Applied in immunofluorescent (IF) and immunohistochemical (IHC) assays, this antibody and rabbit polyclonal antibody could give the consistent results in SH-SY5Y cells, human sperm, and mouse brain paraffin section. In summary, we generate a mouse InsP3R1-specific IgG 2b antibody identifying InsP3R1 in WB, IF, IHC, and IP analysis, which provides a possible choice for detection of InsP3R1, especially in application of co-localization analysis with other InsP3R isoforms or other proteins.

Combined 3D DNA FISH, Single-Molecule RNA FISH, and Immunofluorescence.

Nuclear architecture is a potential regulator of gene expression in eukaryotic cells. Studies connecting nuclear architecture to gene expression are often population-averaged and do not report on the cell-level heterogeneity in genome organization and associated gene expression. In this report we present a simple way to combine fluorescence in situ hybridization (FISH)-based detection of DNA, with single-molecule RNA FISH (smFISH) and immunofluorescence (IF), while also preserving the three-dimensional (3D) nuclear architecture of a cell. Recently developed smFISH techniques enable the detection of individual RNA molecules; while using 3D DNA FISH, copy numbers and positions of genes inside the nucleus can be interrogated without interfering with 3D nuclear architecture. Our method to combine 3D DNA FISH with smFISH and IF enables a unique quantitative handle on the central dogma of molecular biology.

Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence.

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19.

Immunohistochemistry and Immunofluorescence.

Immunohistochemistry (IHC) is one of the most widely used protein detection techniques. The principle of this technique is based on the binding of a specific antibody to a matching specific antigen in tissue. The bound antigen-antibody complex then is visualized using a range of detection techniques. IHC uses a number of different enzymatic labels, such as peroxidase and alkaline phosphatase, for the detection of the antigens of interest whereas immunofluorescence (IF) uses a fluorescent signal. In this chapter, IHC will be described using the peroxidase label. Both IHC and IF can be used on formalin-fixed paraffin-embedded (FFPE) or appropriately processed fresh tissues. IHC/IF can be multiplexed to detect more than one antigen at a time, or may be sequentially stained to detect multiple targets. These techniques are routinely used in diagnostic pathology laboratories, not just for diagnostic purposes but many biomarkers are used for patient staging, treatment allocation, and prognostication. Immunofluorescence is routinely used for the detection of antibodies and antigens in freshly biopsied tissues, particularly for immune-mediated and vesiculobullous lesions. In this chapter, the principles of IHC are reviewed followed by examples of IHC and IF staining using readily available antibodies. Steps and processes involved in IHC/IF double staining are also described.

Multiplex Spatial Protein Detection by Combining Immunofluorescence with Immunohistochemistry.

Technologies for staining and imaging multiple antigens in single tissue sections are developing rapidly due to their potential to uncover spatial relationships between proteins with cellular resolution. Detections are performed simultaneously or sequentially depending on the approach. However, several technologies can detect limited numbers of antigens or require expensive equipment and reagents. Another serious concern is the lack of flexibility. Most commercialized reagents are validated for defined antibody panels, and introducing any changes is laborious and costly. In this chapter, we describe a method where we combine, for the first time, multiplexed IF followed by sequential immunohistochemistry (IHC) with AEC chromogen on Leica Bond staining processors with paraffin tissue sections. We present data for successful detection of 10 antigens in a single tissue section with preserved tissue integrity. Our method is designed for use with any combination of antibodies of interest, with images collected using whole slide scanners. We include an image viewing and image analysis workflow using nonlinear warping to combine all staining passes in a single full-resolution image of the entire tissue section, aligned at the single cell level.

Multiplex Fluorescent Immunohistochemistry for Preservation of Tumor Microenvironment Architecture and Spatial Relationship of Cells in Tumor Tissues.

The tumor microenvironment (TME), composed of immune cells, antigens, and local soluble factors, is integral to cancer development and progression. Traditional techniques such as immunohistochemistry, immunofluorescence, or flow cytometry limit the analysis of spatial data and cellular interactions within the TME, as they are restricted to colocalization of a small number of antigens or the loss of tissue architecture. Multiplex fluorescent immunohistochemistry (mfIHC) allows for detection of multiple antigens within a single tissue sample, providing a more comprehensive description of tissue composition and spatial interactions within the TME. This technique utilizes antigen retrieval, application of primary and secondary antibodies, followed by a tyramide-based chemical reaction to covalently bind a fluorophore to an epitope of interest and, eventually, stripping of the antibodies. This allows for multiple rounds of antibody application without concern for species cross-reactivity, as well as signal amplification which abrogates the autofluorescence that frequently plagues analysis of fixed tissues. As such, mfIHC can be used to quantify multiple cellular populations and their interactions, in situ, unlocking key biologic data that was previously unavailable. This chapter provides an overview of the experimental design, staining, and imaging strategies using a manual technique in formalin-fixed paraffin-embedded tissue sections.

CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer.

Androgen deprivation therapy (ADT) is the main treatment for advanced prostate cancer (PCa) but resistance results in progression to terminal castrate resistant PCa (CRPC), where there is an unmet therapeutic need. Aberrant intracellular calcium (Cai2+) is known to promote neoplastic transformation and treatment resistance. There is growing evidence that voltage gated calcium channel (VGCC) expression is increased in cancer, particularly CACNA1D/CaV1.3 in CRPC. The aim of this study was to investigate if increased CaV1.3 drives resistance to ADT and determine its associated impact on Cai2+ and cancer biology. Bioinformatic analysis revealed that CACNA1D gene expression is increased in ADT treated PCa patients. This was corroborated in both in vivo LNCaP xenograft mouse and in vitro PCa cell line models, which demonstrated a significant increase in CaV1.3 protein expression following ADT with bicalutamide. Expression was found to be of a shortened 170kDa CaV1.3 isoform associated with plasma and intracellular membranes, which failed to induce calcium influx following membrane depolarisation. Instead, under ADT CaV1.3 mediated a rise in basal cytosolic calcium and an increase in store operated calcium entry (SOCE). This mechanism was found to promote the proliferation and survival of ADT resistant CRPC cells. Overall, this study demonstrates for the first time in PCa that under ADT specific CaV1.3 isoforms promote an upregulation of SOCE which contributes to treatment resistance and CRPC biology. Thus, this novel oncochannel represents a target for therapeutic development to improve PCa patient outcomes.

An improved and simplified protocol to combine Golgi-Cox staining with immunofluorescence and transmission electron microscopy techniques.

Approaches utilizing multiple analysis techniques on a single sample are highly desirable in research, especially to reduce the number of animals and obtain the maximum information. Golgi-Cox staining is a widely used method for characterizing axon and dendritic morphology and several attempts to combine this technique with immunofluorescence and transmission electron microscopy have been proposed. With few exceptions, most of the protocols were characterized by a high degree of complexity and low reproducibility. Here we show a simplified procedure of perfusion, fixation and staining of brain tissues that allows Golgi-Cox staining, immunofluorescence and transmission electron microscopy in the same sample, to obtain high-quality images with a low-cost procedure. The main novelty in this protocol is the possibility of performing Golgi-Cox staining after the perfusion and post-fixation of brain tissue with a buffered solution containing, not only formaldehyde, but also glutaraldehyde. This renders the tissue suitable for electron microscopy, but it is also compatible with immunofluorescence staining. This combined protocol can be used in most neuroscience laboratories as it does not require special equipment and skills. This protocol will be useful in a broad range of neuroscience topics to study morphological changes during brain development and plasticity in physiological and pathological conditions.

Complement Detection in Human Tumors by Immunohistochemistry and Immunofluorescence.

Tumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of the cells. There is a high heterogeneity of the staining within tumors but also between patients. Semi-quantification of the staining in large cohorts of patients allows to investigate the prognostic impact of the local complement production and activation. Here we explain the staining process for C1q, C4, and C3 in human paraffin-embedded tumor sections by immunofluorescence and immunohistochemistry.

Methods to Detect NF-κB Activity in Tumor-Associated Macrophage (TAM) Populations.

Macrophages are an abundant population in the tumor-infiltrating immune cells. The transcription factor NF-κB plays an important role in the response of tumor-associated macrophages (TAMs) to the tumor environmental cues. Detecting NF-κB activity in TAMs will help define the functional status of the TAMs. In this article, we describe several methods to detect NF-κB activity in TAM populations.

Fluorescence In Situ Hybridization (FISH) Detection of Viral Nucleic Acids in Oncolytic H-1 Parvovirus-Treated Human Brain Tumors.

Fluorescence in situ hybridization (FISH) is a specific, sensitive, accurate, and reliable technique widely applied in both research and clinic. Here we describe the detailed protocol of a FISH method established by us to serve the scientific purposes of the first oncolytic parvovirus clinical trial (ParvOryx01). This trial was launched in Germany in 2011. After trial completion in 2015, results were published in Molecular Therapy in 2017. The primary purpose of the trial was to evaluate the safety of an oncolytic parvovirus, H-1PV (ParvOryx), in recurrent glioblastoma patients. In addition, the efficiency of H-1PV tumor targeting after intratumoral or systemic virus administration was assessed by FISH detection of viral nucleic acids (genomic single-stranded DNA, mRNA and parvovirus double-stranded replicative forms) in formalin-fixed paraffin-embedded glioblastoma tissues resected at day 10 after ParvOryx treatment. The FISH method allowed the detection-for the first time in humans-of H-1PV replication markers in brain tumors of parvovirus-treated patients. A protocol combining mRNA FISH with simultaneous immunofluorescent staining for tumor and tumor microenvironment markers was also developed and is described here, in order to better characterize H-1PV cellular targets and H-1PV treatment-associated tumor microenvironment changes.

A new ligand of CD105 screened out by phage display technology provides a reliable identification of recurrent or metastasizing pleomorphic adenoma from pleomorphic adenoma.

OBJECTIVES: To assess CD105 expression in pleomorphic adenoma (PA), recurrent pleomorphic adenoma (RPA) and metastasizing pleomorphic adenoma (MPA), to identify new epitopes and screen a ligand with high affinity to CD105 by phage display technology, to evaluate the reliability of the new ligand for identifying RPA/MPA from PA. METHODS: Phage display technology was used to screen ligands with high affinity to recombinant human CD105. The ligand with strongest affinity to CD105 was synthesized by FMOC Chemistry according to the sequencing results. The archived formalin fixed paraffin-embedded (FFPE) tissues of 35 PA cases, 12 RPA cases and 2 MPA cases were sliced and immunofluorescent stained. CD105 expression were detected by Confocal laser scanning microscopy (CLSM). The relative fluorescence intensity was calculated with the image processing software Image J. Statistical analyses were performed by the software Graph Pad Prism (Version 7.0a). Using PROC logistic, receiver operating characteristic (ROC) curves, area under ROC curves (AUCs) were generated to assess the sensitivity and specificity of the new ligand for identifying RPA/MPA from PA cases. RESULTS: A ligand with specialty and high affinity to CD105 i.e. ligand nABPK296 were developed. FITC-labeled ligand nABPK296 confirmed the difference of CD105 expression in RPA/MPA and PA. The AUC of nABPK296 was 0.9418. CONCLUSIONS: CD105 is a promising biomarker for identification of RPA/MPA from PA cases. Ligand nABPK296 provides a promising approach to CD105 detection. This study also validated the reliability of phage display technology in finding new episodes and ligands with high affinity for antigens.

Immunohistochemical profiling of estrogen-related receptor gamma in rat brain and colocalization with estrogen receptor alpha in the preoptic area.

Estrogen-related receptor (ERR) is a member of the nuclear receptor superfamily that has strong homology with estrogen receptor (ER) α. Despite the lack of endogenous ligands, ERR serves as transcription factors through their constitutively active structure with or without interaction with ERα. Among the three subtypes of ERR (α, ß, and γ), ERRγ is highly expressed in brain, but the distribution of ERRγ is poorly characterized. Therefore, we investigated ERRγ immunoreactivity throughout the rostro-caudal axis in rat brain. Immunohistochemistry revealed localization of ERRγ protein in the cell nucleus, and a ubiquitous distribution of ERRγ in brain regions including the olfactory bulb, cerebrum, brain stem, and cerebellum. Selective intense immunoreactivity was observed in the reticular thalamic nucleus, zona incerta, circular nucleus, interpeduncular nucleus, pontine nucleus, and parasolitary nucleus. Most ERRγ-immunoreactive (ir) regions were also positive for ERα and/or ERß, which suggests that ERRγ is involved in modulation of estrogen signaling in adult rat brain. Double immunofluorescence demonstrated colocalization of ERRγ with ERα within the anteroventral periventricular nucleus of the preoptic area (AVPV) and medial preoptic nucleus (MPO), which are major target sites for estrogen action. The results of this study suggest that ERRγ function in the brain is affected by estrogens through an interaction with ERα. The findings also provide basic information on brain region-specific ERRγ function.

Broad-spectrum protein kinase inhibition by the staurosporine analog KT-5720 reverses ethanol withdrawal-associated loss of NeuN/Fox-3.

Chronic, intermittent ethanol (CIE) exposure is known to produce neuroadaptive alterations in excitatory neurotransmission that contribute to the development of dependence. Although activation of protein kinases (e.g., cyclic AMP [cAMP]-dependent protein kinase) is implicated in the synaptic trafficking of these receptors following CIE exposure, the functional consequences of these effects are yet to be fully understood. The present study sought to delineate the influence of protein kinase in regulating cytotoxicity following CIE exposure, as well as to examine the relative roles of ethanol exposure and ethanol withdrawal (EWD) in promoting these effects. Rat hippocampal explants were exposed to a developmental model of CIE with or without co-application of broad-spectrum protein kinase inhibitor KT-5720 (1 µM) either during ethanol exposure or EWD. Hippocampal cytotoxicity was assessed via immunofluorescence (IF) of neuron-specific nuclear protein (NeuN) with thionine staining of Nissl bodies to confirm IF findings. Concomitant application of ethanol and KT-5720 restored the loss of NeuN/Fox-3 IF in pyramidal CA1 and granule DG cell layers produced by CIE, but there was no restoration in CA3. Application of KT-5720 during EWD failed to significantly alter levels of NeuN IF, implying that ethanol exposure activates protein kinases that, in part, mediate the effects of EWD. KT-5720 application during EWD also restored thionine staining in CA1, suggesting kinase regulation of both neurons and non-neuronal cells. These data demonstrate that CIE exposure alters protein kinase activity to promote ethanol withdrawal-associated loss of NeuN/Fox-3 and highlight the influence of kinase signaling on distinct cell types in the developing hippocampus.

Automated Double In Situ Detection of Mouse Lgr5 mRNA and Lysozyme Protein in Examining the Neighboring Cell Types of the Mouse Intestinal Crypt.

Automated detection of mRNAs and proteins in the same tissue sections is not a routine procedure. Successful experiment depends on the preparation of the tissue, the detection procedure, as well as the quality of the probes and antibodies. The multiplexed detections require experimental conditions, preserving the state of the molecular targets of interest and providing expression pattern of each target the same as in a single detection. Here we describe in detail the automated protocols used to detect mouse Lgr5 mRNA by in situ hybridization and immunofluorescence detection of lysozyme in the same mouse intestinal sections. Both the in situ hybridization and the protein detection were performed with an automated staining processor and provided strong and reproducible results.

Microfluidic Capture and Multiplex Immunofluorescence of Circulating Tumor Cells to Identify Cancer of Origin.

Circulating tumor cells (CTCs) are an important biomarker and their analysis can be considered a form of "liquid biopsy." The purpose of this book chapter is to describe the use of the 4-channel CMx (cells captured in maximum) microfluidic chip, containing special micropatterns coated with an antibody-conjugated supported lipid bilayer (SLB) on its surface, to capture and isolate CTCs from the blood of cancer patients. Captured CTCs are subsequently released by an air foam to an immunofluorescence (IF) staining panel that enables further analysis, including the identification of the primary cancer source of the CTCs.