Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging.

The tumor microenvironment plays a crucial role in determining response to treatment. This involves a series of interconnected changes in the cellular landscape, spatial organization, and extracellular matrix composition. However, assessing these alterations simultaneously is challenging from a spatial perspective, due to the limitations of current high-dimensional imaging techniques and the extent of intratumoral heterogeneity over large lesion areas. In this study, we introduce a spatial proteomic workflow termed Hyperplexed Immunofluorescence Imaging (HIFI) that overcomes these limitations. HIFI allows for the simultaneous analysis of > 45 markers in fragile tissue sections at high magnification, using a cost-effective high-throughput workflow. We integrate HIFI with machine learning feature detection, graph-based network analysis, and cluster-based neighborhood analysis to analyze the microenvironment response to radiation therapy in a preclinical model of glioblastoma, and compare this response to a mouse model of breast-to-brain metastasis. Here we show that glioblastomas undergo extensive spatial reorganization of immune cell populations and structural architecture in response to treatment, while brain metastases show no comparable reorganization. Our integrated spatial analyses reveal highly divergent responses to radiation therapy between brain tumor models, despite equivalent radiotherapy benefit.

MIM-CyCIF: masked imaging modeling for enhancing cyclic immunofluorescence (CyCIF) with panel reduction and imputation.

Cyclic Immunofluorescence (CyCIF) can quantify multiple biomarkers, but panel capacity is limited by technical challenges. We propose a computational panel reduction approach that can impute the information content from 25 markers using only 9 markers, learning co-expression and morphological patterns while concurrently increasing speed and panel content and decreasing cost. We demonstrate strong correlations in predictions and generalizability across breast and colorectal cancer, illustrating applicability of our approach to diverse tissue types.

Comparative Performance Assessment of Novel Fluorescence Immunoassay POCTs for Measuring Circulating Levels of Vitamin-D.

Vitamin D (Vit D) is a fat-soluble molecule acting like a hormone, and it is involved in several biological mechanisms such as gene expression, calcium homeostasis, bone metabolism, immune modulation, viral protection, and neuromuscular functions. Vit D deficiency can lead to chronic hypocalcemia, hyperparathyroidism, and many other pathological conditions; in this context, low and very low levels of 25-hydroxy-vitamin D (25-OH-D) were found to be associated with an increased risk of COVID-19 infection and the likelihood of many severe diseases. For all these reasons, it is important to quantify and monitor 25-OH-D levels to ensure that the serum/blood concentrations are not clinically suboptimal. Serum concentration of 25-OH-D is currently the main indicator of Vit D status, and it is currently performed by different assays, but the most common quantitation techniques involve immunometric methods or chromatography. Nevertheless, other quantitation techniques and instruments are now emerging, such as AFIAS-1® and AFIAS-10® (Boditech and Menarini) based on the immunofluorescence analyzer, that guarantee an automated system with cartridges able to give quick and reliable results as a point-of-care test (POCT). This work aims to compare AFIAS-1® and AFIAS-10® (Boditech and Menarini) Vit D quantitation with Ultra High-Performance Liquid Chromatography coupled with tandem mass spectrometry that currently represents the gold standard technique for Vit D quantitation. The analyses were performed in parallel on 56 samples and in different conditions (from fresh and frozen plasma) to assess the reliability of the results. Any statistically significant differences in methods, the fixed error, and the error proportional to concentration were reported. Results obtained in all conditions showed a good correlation between both AFIAS® instruments and LC-MS/MS, and we can affirm that AFIAS-1® and AFIAS-10® are reliable instruments for measuring 25-OH-D with accuracy and in a fast manner.

Comparison of the Capacity of Several Machine Learning Tools to Assist Immunofluorescence-Based Detection of Anti-Neutrophil Cytoplasmic Antibodies.

The success of artificial intelligence and machine learning is an incentive to develop new algorithms to increase the rapidity and reliability of medical diagnosis. Here we compared different strategies aimed at processing microscope images used to detect anti-neutrophil cytoplasmic antibodies, an important vasculitis marker: (i) basic classifier methods (logistic regression, k-nearest neighbors and decision tree) were used to process custom-made indices derived from immunofluorescence images yielded by 137 sera. (ii) These methods were combined with dimensional reduction to analyze 1733 individual cell images. (iii) More complex models based on neural networks were used to analyze the same dataset. The efficiency of discriminating between positive and negative samples and different fluorescence patterns was quantified with Rand-type accuracy index, kappa index and ROC curve. It is concluded that basic models trained on a limited dataset allowed for positive/negative discrimination with an efficiency comparable to that obtained by conventional analysis performed by humans (0.84 kappa score). More extensive datasets and more sophisticated models may be required for efficient discrimination between fluorescence patterns generated by different auto-antibody species.

Obtaining Tissues of Human Amniotic Membrane and Identification of Pluripotent Markers.

The immunofluorescence technique has been used to identify pluripotent markers in the human amniotic epithelial cells (hAEC). hAEC belonging to human fetal membranes, specificamently to amnion layer, and are arising by epiblast, this sugest that the hAEC have characteristics of epiblast cells, in other words, characteristcs of pluripotent stem cells. Here we describe obtaining human amnion tissue and identifying pluripotent markers by immunofluorescence.

RNA and Protein Detection by Single-Molecule Fluorescent in Situ Hybridization (smFISH) Combined with Immunofluorescence in the Budding Yeast S. cerevisiae.

The inherent stochastic processes governing gene expression give rise to heterogeneity across individual cells, highlighting the importance of single-cell studies. The emergence of single-molecule fluorescent in situ hybridization (smFISH) enabled gene expression analysis at the single-cell level while including the spatial dimension through the visualization and quantification of mRNAs in intact fixed cells. By combining smFISH with immunofluorescence (IF), a comprehensive approach takes shape facilitating the study of mRNAs and proteins to correlate gene expression profiles to different cellular states. This chapter serves as a comprehensive guide to a smFISH-IF protocol optimized for gene expression analysis in the budding yeast S. cerevisiae. We utilize smFISH to visualize the mRNA localization pattern of the CLB2 cyclin over the course of the cell cycle inferred by alpha-tubulin IF.

Multiplexed Immunofluorescence and Single-Molecule RNA Fluorescence In Situ Hybridization in Mouse Skeletal Myofibers.

RNA fluorescence in situ hybridization (FISH) is a powerful method to determine the abundance and localization of mRNA molecules in cells. While modern RNA FISH techniques allow quantification at single molecule resolution, most methods are optimized for mammalian cell culture and are not easily applied to in vivo tissue settings. Single-molecule RNA detection in skeletal muscle cells has been particularly challenging due to the thickness and high autofluorescence of adult muscle tissue and a lack of in vitro models for mature muscle cells (myofibers). Here, we present a method for isolation of adult myofibers from mouse skeletal muscle and detection of single mRNA molecules and proteins using multiplexed RNA FISH and immunofluorescence.

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.

Cell-Based Assay to Detect the Autoantibody Serostatus in Patients with Neuromyelitis Optica Spectrum Disorder (NMOSD).

Cell-based assay (CBA) is an immunofluorescence assay that is extensively used for the confirmatory diagnosis of inflammatory demyelinating diseases of the central nervous system, like neuromyelitis optica spectrum disorder (NMOSD). Detecting the type of autoantibody present in the sera of the patients is the primary goal. CBA is the most sensitive and recommended detection method among all similar tools. Briefly, serum autoantibody is screened by transfecting specific cells seeded on cover glasses with full-length specific antigen fused with green fluorescent protein (GFP), followed by treating them with the patient serum used here as the source of primary antibody. The autoantibody-treated cells are further labeled with a rhodamine-conjugated secondary antibody. The co-localization of GFP and rhodamine is visualized by confocal microscopy, and the intensity of fluorescence is evaluated to determine the presence of autoantibody. A detailed protocol to screen antibodies against AQP4 and MOG in human sera using this method is described.

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.

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Multiplex Immunofluorescence Staining Protocol for the Dual Imaging of Hypoxia-Inducible Factors 1 and 2 on Formalin-Fixed Paraffin-Embedded Samples.

Hypoxia is a common condition in rapidly proliferating tumors and occurs when oxygen delivery to the tissue is scarce. It is a prevalent feature in ~90% of solid tumors. The family of HIF (hypoxia-inducible factor) proteins-HIF1α and HIF2α-are the main transcription factors that regulate the response to hypoxia. These transcription factors regulate numerous downstream gene targets that promote the aggressiveness of tumors and therefore have been linked to worse prognosis in patients. This makes them a potential biomarker to be tested in the clinical setting to predict patient outcomes. However, HIFs have been notoriously challenging to immunolabel, in part due to their fast turnover under normal oxygen conditions. In this work, we developed a multiplexed immunofluorescence (mIF) staining protocol for the simultaneous detection of HIF1α and HIF2α in the same formalin-fixed paraffin-embedded (FFPE) tissue section.

Cell Cycle Mapping Using Multiplexed Immunofluorescence.

The development of technologies that allow measurement of the cell cycle at the single-cell level has revealed novel insights into the mechanisms that regulate cell cycle commitment and progression through DNA replication and cell division. These studies have also provided evidence of heterogeneity in cell cycle regulation among individual cells, even within a genetically identical population. Cell cycle mapping combines highly multiplexed imaging with manifold learning to visualize the diversity of "paths" that cells can take through the proliferative cell cycle or into various states of cell cycle arrest. In this chapter, we describe a general protocol of the experimental and computational components of cell cycle mapping. We also provide a comprehensive guide for the design and analysis of experiments, discussing key considerations in detail (e.g., antibody library preparation, analysis strategies, etc.) that may vary depending on the research question being addressed.

Efficient 3D imaging and pathological analysis of the human lymphoma tumor microenvironment using light-sheet immunofluorescence microscopy.

Rationale: The composition and spatial structure of the lymphoma tumor microenvironment (TME) provide key pathological insights for tumor survival and growth, invasion and metastasis, and resistance to immunotherapy. However, the 3D lymphoma TME has not been well studied owing to the limitations of current imaging techniques. In this work, we take full advantage of a series of new techniques to enable the first 3D TME study in intact lymphoma tissue. Methods: Diverse cell subtypes in lymphoma tissues were tagged using a multiplex immunofluorescence labeling technique. To optically clarify the entire tissue, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+), clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) and stabilization to harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD) were comprehensively compared with the ultimate dimensional imaging of solvent-cleared organs (uDISCO) approach selected for clearing lymphoma tissues. A Bessel-beam light-sheet fluorescence microscope (B-LSFM) was developed to three-dimensionally image the clarified tissues at high speed and high resolution. A customized MATLAB program was used to quantify the number and colocalization of the cell subtypes based on the acquired multichannel 3D images. By combining these cutting-edge methods, we successfully carried out high-efficiency 3D visualization and high-content cellular analyses of the lymphoma TME. Results: Several antibodies, including CD3, CD8, CD20, CD68, CD163, CD14, CD15, FOXP3 and Ki67, were screened for labeling the TME in lymphoma tumors. The 3D imaging results of the TME from three types of lymphoma, reactive lymphocytic hyperplasia (RLN), diffuse large B-cell lymphoma (DLBCL), and angioimmunoblastic T-cell lymphoma (AITL), were quantitatively analyzed, and their cell number, localization, and spatial correlation were comprehensively revealed. Conclusion: We present an advanced imaging-based method for efficient 3D visualization and high-content cellular analysis of the lymphoma TME, rendering it a valuable tool for tumor pathological diagnosis and other clinical research.

Evaluating the State of Glomerular Disease by Analyzing Urinary Sediments: mRNA Levels and Immunofluorescence Staining for Various Markers.

Renal biopsy is the gold standard for making the final diagnosis and for predicting the progression of renal disease, but monitoring disease status by performing biopsies repeatedly is impossible because it is an invasive procedure. Urine tests are non-invasive and may reflect the general condition of the whole kidney better than renal biopsy results. We therefore investigated the diagnostic value of extensive urinary sediment analysis by immunofluorescence staining for markers expressed on kidney-derived cells (cytokeratin: marker for tubular epithelial cells, synaptopodin: marker for podocytes, claudin1: marker for parietal epithelial cells, CD68: marker for macrophages (MΦ), neutrophil elastase: marker for neutrophils). We further examined the expression levels of the mRNAs for these markers by real-time reverse transcription polymerase chain reaction. We also examined the levels of mRNAs associated with the M1 (iNOS, IL-6) and M2 (CD163, CD204, CD206, IL-10) MΦ phenotypes. Evaluated markers were compared with clinical and histological findings for the assessment of renal diseases. Claudin1- and CD68-positive cell counts in urinary sediments were higher in patients with glomerular crescents (especially cellular crescents) than in patients without crescents. The relative levels of mRNA for CD68 and the M2 MΦ markers (CD163, CD204, CD206, and IL-10) in urinary sediments were also higher in patients with glomerular crescents. These data suggest that immunofluorescence staining for claudin1 and CD68 in urinary sediments and the relative levels of mRNA for CD68 and M2 MΦ markers in urinary sediments are useful for evaluating the state of glomerular diseases.

Immunofluorescence analysis of human eosinophils.

A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the immune response towards an allergic phenotype. Unfortunately, it is challenging to immunolabel and collect quantifiable images of eosinophils given their innate autofluorescence and ability to nonspecifically bind to antibodies. Hence, it is important to optimize permeabilization, blocking, and imaging conditions for eosinophils. Here, we show enhanced protocols to ensure that measured immunofluorescence represents specific immunolabelling. To test this, eosinophils were purified from human blood, adhered to glass coverslips, stimulated with or without platelet-activating factor (PAF), fixed with paraformaldehyde, and then permeabilized with Triton X-100 or saponin. Cells were then blocked with goat serum or human serum and incubated with antibodies labelling cytokines (IL-9 and IL-13) and secretory organelles (CD63 for crystalloid granules and transferrin receptor [TfnRc] for recycling endosomes). Carefully selected isotype controls were used throughout, and cells were imaged using Deltavision super-resolution microscopy. Intensities of fluorescent probes were quantified using Volocity software. Our findings show that permeabilization with saponin, blockage with human serum, and using concentrations of antibodies up to 10 µg/ml allowed us to detect marked differences in fluorescence intensities between isotypes and test antibodies. With the achievement of sufficient qualitative and quantitative measures of increased test probe intensity compared to respective isotypes, these results indicate that our protocol allows for optimal immunolabelling of eosinophils. Using this protocol, future studies may provide further insights into trafficking mechanisms within this important inflammatory cell type.

Whole-Mount Immunohistochemical and Immunofluorescence Assays in Zebrafish Embryos.

Zebrafish embryos are an important organism used as an in vivo model in a wide variety of disciplines from the past to the present. Immunohistochemistry analyses are an important method used to determine the localization of specific antigens in tissue sections with labeled antibodies depending on antigen-antibody interactions in zebrafish embryos. Immunofluorescence assays are an immunohistochemistry method that uses fluorophores to determine diverse cellular antigens. Zebrafish embryos and larvae, with their small size, are the most ideal model organisms for whole-mount immunohistochemical and immunofluorescent methods today. The small size of these organisms allows simultaneous evaluation of different tissues and organs, and results are obtained in a shorter time. In this section, whole-mount immunohistochemical and immunofluorescent analysis methods in zebrafish embryos, and larvae are summarized in detail, taking into account different studies and recent advances.

Data Science for Health Image Alignment: A User-Friendly Open-Source ImageJ/Fiji Plugin for Aligning Multimodality/Immunohistochemistry/Immunofluorescence 2D Microscopy Images.

Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle's principle, "The whole is greater than the sum of its parts", multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available.

Combined amplification-based single-molecule fluorescence in situ hybridization with immunofluorescence for simultaneous in situ detection of RNAs and proteins.

We present a combined amplification-based single-molecule fluorescence in situ hybridization and immunofluorescence (asmFISH-IF) method for the detection of multiple RNAs and proteins simultaneously in cells and formaldehyde-fixed and paraffin-embedded tissue sections. We showed that performing asmFISH before immunofluorescence gives a better IF signal than the opposite. Our asmFISH-IF method could help study the interplay of RNA and protein, helping to understand their functions.