{"id":5042,"date":"2025-10-08T18:13:09","date_gmt":"2025-10-08T18:13:09","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/life-in-the-lab\/?p=5042"},"modified":"2025-10-08T18:15:15","modified_gmt":"2025-10-08T18:15:15","slug":"spatial-omics-dual-ish-ihc-brain-mapping","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/life-in-the-lab\/spatial-omics-dual-ish-ihc-brain-mapping\/","title":{"rendered":"Spatial Omics Meets Neuroscience: Dual ISH-IHC Brain Mapping Case Study"},"content":{"rendered":"\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"921\" height=\"601\" src=\"https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/Spatial-Bio-Image.jpg\" alt=\"Spatial Biology Meets Neuroscience: Dual ISH-IHC Brain Mapping\" class=\"wp-image-5043\" srcset=\"https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/Spatial-Bio-Image.jpg 921w, https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/Spatial-Bio-Image-300x196.jpg 300w, https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/Spatial-Bio-Image-768x501.jpg 768w\" sizes=\"auto, (max-width: 921px) 100vw, 921px\" \/><\/figure>\n\n\n\n<div style=\"height:25px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>Spatial omics biology is reshaping how researchers study complex tissues by preserving the molecular context that traditional methods often lose.<\/p>\n\n\n\n<p>For scientists pushing toward true spatial multi-omics, integrating immunohistochemistry (IHC) with in situ hybridization (ISH) represents both an opportunity and a technical challenge.<\/p>\n\n\n\n<p>This blog explores a recent application note that demonstrates how careful protocol modifications unlock dual RNA\u2013protein detection in the same tissue section\u2014showing what\u2019s possible when spatial biology moves from theory to practice.<\/p>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<div class=\"wp-block-yoast-seo-table-of-contents yoast-table-of-contents\"><h2>Table of contents<\/h2><ul><li><a href=\"#h-what-is-spatial-biology-and-multi-omics\" data-level=\"2\">What is spatial biology and multi-omics?<\/a><ul><li><a href=\"#h-what-is-spatial-proteomics\" data-level=\"3\">What is spatial proteomics?<\/a><\/li><li><a href=\"#h-what-is-spatial-transcriptomics\" data-level=\"3\">What is spatial transcriptomics?<\/a><\/li><li><a href=\"#h-what-is-spatial-multi-omics\" data-level=\"3\">What is spatial multi-omics?<\/a><\/li><\/ul><\/li><li><a href=\"#h-getting-multi-omics-right-with-multiplexing-nbsp\" data-level=\"2\">Getting multi-omics right with multiplexing\u00a0<\/a><ul><li><a href=\"#h-ihc-vs-ish-different-targets-different-chemistry\" data-level=\"3\">IHC vs ISH: Different targets, different chemistry<\/a><\/li><li><a href=\"#h-combining-ihc-and-ish\" data-level=\"3\">Combining IHC and ISH<\/a><\/li><\/ul><\/li><li><a href=\"#h-spatial-multi-omics-in-action-brain-mapping-in-mice\" data-level=\"2\">Spatial multi-omics in action: brain mapping in mice<\/a><ul><li><a href=\"#h-approach\" data-level=\"3\">Approach<\/a><\/li><li><a href=\"#h-results\" data-level=\"3\">Results<\/a><\/li><\/ul><\/li><li><a href=\"#h-spatial-biology-and-multi-omics-technologies\" data-level=\"2\">Spatial biology and multi-omics technologies<\/a><\/li><li><a href=\"#h-imaging-platforms\" data-level=\"2\">Imaging platforms<\/a><ul><li><a href=\"#h-rna-detection\" data-level=\"3\">RNA detection<\/a><\/li><li><a href=\"#h-protein-detection\" data-level=\"3\">Protein detection<\/a><\/li><\/ul><\/li><li><a href=\"#h-key-takeaways\" data-level=\"2\">Key takeaways<\/a><\/li><li><a href=\"#h-more-spatial-biology-and-multi-omics-resources\" data-level=\"2\">More spatial biology and multi-omics resources<\/a><\/li><\/ul><\/div>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<div class=\"wp-block-buttons is-content-justification-center is-layout-flex wp-container-core-buttons-is-layout-16018d1d wp-block-buttons-is-layout-flex\">\n<div class=\"wp-block-button has-custom-width wp-block-button__width-75 is-style-fill\"><a class=\"wp-block-button__link has-white-color has-text-color has-background has-link-color has-medium-font-size has-text-align-center has-custom-font-size wp-element-button\" href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/in-situ-hybridization-ish\/rna-fish\/viewrna-assays.html\" style=\"border-radius:0px;background-color:#ee3134\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Explore Invitrogen ViewRNA ISH assays<\/strong><\/a><\/div>\n<\/div>\n\n\n\n<div style=\"height:25px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"h-what-is-spatial-biology-and-multi-omics\">What is spatial biology and multi-omics?<\/h2>\n\n\n\n<p>Spatial biology represents a significant advancement in our understanding of biological systems.<\/p>\n\n\n\n<p>Many molecular analysis techniques strip away one crucial piece of information: spatial context. That is, where molecules sit and how they interact in the wider tissue environment.<\/p>\n\n\n\n<p>Spatial biology preserves this key context. Rather than grinding up tissue samples and losing all positional data, researchers using spatial biology techniques can maintain the structural integrity of specimens. This allows them to visualize and quantify proteins, RNA, and other biomolecules exactly where cells produce and use them.<\/p>\n\n\n\n<p>For neuroscientists studying brain architecture, this spatial context makes all the difference between understanding isolated molecular events and grasping how those events coordinate across complex neural networks.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-what-is-spatial-proteomics\">What is spatial proteomics? <\/h3>\n\n\n\n<p>Proteomics aims to identify, characterize and quantify all proteins (the proteome) in a given system like a cell, tissue, or organism.<\/p>\n\n\n\n<p>Unlike the relatively static genome, the proteome is highly dynamic.&nbsp; Proteins change in their abundance, structure, post-translational modification profile, and environmental response all the time. Proteomic approaches can help researchers bridge the gap between genotype and phenotype to better understand cellular function, disease mechanisms, biomarkers, and more.<\/p>\n\n\n\n<p>Traditional proteomics workflows involve extracting proteins from homogenized tissue samples. While this approach can help identify and quantify proteins with high precision, it destroys valuable spatial information about protein localization patterns.<\/p>\n\n\n\n<p>Spatial proteomics considers protein distribution, abundance, and interaction within the natural tissue environment. Instead of asking simply, <em>&#8220;what proteins are present,&#8221;<\/em> spatial proteomics asks, <em>&#8220;where are these proteins located, and what does that tell us about cellular function?&#8221;<\/em><\/p>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-what-is-spatial-transcriptomics\">What is spatial transcriptomics?<\/h3>\n\n\n\n<p>Transcriptomics is the study of the transcriptome, or the complete set of RNA transcripts in a system.<\/p>\n\n\n\n<p>Spatial transcriptomics approaches can reveal how gene expression varies across different regions of a tissue sample, providing insights into cellular heterogeneity and tissue architecture that would be lost in conventional bulk RNA sequencing methods.<\/p>\n\n\n\n<p>Spatial transcriptomics might look like a researcher working with brain tissue and identifying region-specific gene expression patterns that are associated with neural function, development, or pathology.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-what-is-spatial-multi-omics\">What is spatial multi-omics?<\/h3>\n\n\n\n<p>Spatial multi-omics adds yet another layer of contextualization to our biological understanding by combining multiple spatial \u201comics\u201d datasets \u2013 including spatial transcriptomics and proteomics.<\/p>\n\n\n\n<p>For example, combining spatial transcriptomics with spatial proteomics allows researchers to correlate gene expression patterns with protein abundance and localization in the same tissue section.<\/p>\n\n\n\n<p>Spatial multi-omics is particularly valuable for understanding complex biological systems like the brain, where cellular heterogeneity and regional specialization play crucial roles in function. By simultaneously mapping multiple molecular features across tissue sections, researchers can identify coordinated changes in gene expression and protein abundance that may drive normal development or contribute to disease pathology.<\/p>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"h-getting-multi-omics-right-with-multiplexing-nbsp\">Getting multi-omics right with multiplexing&nbsp;<\/h2>\n\n\n\n<p>Combining spatial proteomics with transcriptomics sounds straightforward in theory. The reality proves more challenging.<\/p>\n\n\n\n<p>To understand why requires a deep dive into two key methods: immunohistochemistry (IHC) and in situ hybridization (ISH).<sub><\/sub><\/p>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-ihc-vs-ish-different-targets-different-chemistry\">IHC vs ISH: Different targets, different chemistry<\/h3>\n\n\n\n<p><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/protein-biology\/protein-biology-learning-center\/protein-biology-resource-library\/pierce-protein-methods\/overview-immunohistochemistry.html\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Immunohistochemistry (IHC)<\/strong><\/a> uses antibodies that bind specifically to protein targets within tissue sections. You can label these antibodies with fluorophores for direct visualization or with enzymes that generate colorimetric signals. After decades of refinement by researchers, IHC offers reliable protein localization through well-validated antibodies.<\/p>\n\n\n\n<p><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/in-situ-hybridization-ish.html\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>In situ hybridization (ISH)<\/strong><\/a> takes a different approach, detecting RNA through complementary probes that hybridize to target sequences. When pairs of probes bind in <a>close proximity<\/a> along the RNA, they create a structure that serves as the foundation for branched DNA amplification. Successive layers of amplifiers and labeled probes build on this scaffold, producing a strong signal with minimal background. This approach enables highly sensitive detection, often at the single-molecule level, while preserving spatial context.<\/p>\n\n\n\n<p><strong>In short: <\/strong>IHC reveals the final protein products of gene expression, while ISH captures the intermediate mRNA transcripts.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-combining-ihc-and-ish\">Combining IHC and ISH<\/h3>\n\n\n\n<p>Successful integration of immunohistochemistry (IHC) with in situ hybridization (ISH) requires confronting a fundamental conflict: optimal conditions for each technique directly oppose each other. IHC antibodies degrade during the protease treatments that ISH requires, while the RNases present during IHC protocols destroy RNA targets needed for ISH detection.<\/p>\n\n\n\n<p>Recent studies have identified specific protocol modifications that address these competing demands. Tissues need to be pretreated with RNase inhibitors before and during IHC labeling to protect RNA integrity. Following IHC labeling, antibodies require crosslinking to the tissue\u2014standard formaldehyde fixation alone cannot withstand the harsh protease treatments necessary for ISH protocols. When executed properly, these modifications enable robust dual detection of both protein and mRNA targets in the same tissue section.<\/p>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><thead><tr><th><strong>Immunohistochemistry (IHC)<\/strong><\/th><th><strong>In Situ Hybridization (ISH)<\/strong><\/th><\/tr><\/thead><tbody><tr><td><strong>What it detects:<\/strong> Proteins (antigens)<br><br><strong>How it works<\/strong>: Antibodies tagged with <br>fluorophores or enzymes bind to target proteins<br><br><strong>Output:<\/strong> Fluorescent or chromogenic protein localization<br><br><strong>Spatial analysis application: <\/strong>Defining cell neighborhoods and tissue architecture<\/td><td><strong>What it detects: <\/strong>RNA transcripts (mRNA, miRNA)<br><br><strong>How it works: <\/strong>Nucleic acid probes hybridize to target RNA; amplified via branched DNA systems<br><strong>Output: <\/strong>Fluorescent or chromogenic transcript localization<br><br><strong>Spatial analysis application: <\/strong>Revealing active transcriptional states and cell heterogeneity<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<p><strong>\u26a0\ufe0f <em>Note: Combining IHC and ISH in the same sample introduces cross-interference.<\/em><\/strong><\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Protease digestion during ISH can destroy antibody epitopes (IHC signal loss).<\/li>\n\n\n\n<li>Antibody reagents can introduce RNases that degrade RNA (ISH signal loss).<\/li>\n\n\n\n<li>Multiplex workflows must include RNase inhibition and antibody crosslinking to preserve both signals.<\/li>\n<\/ul>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"h-spatial-multi-omics-in-action-brain-mapping-in-mice\">Spatial multi-omics in action: brain mapping in mice <\/h2>\n\n\n\n<p>Thermo Fisher Scientific R&amp;D experts recently tackled the challenge of dual IHC-ISH for spatial biology analysis.<\/p>\n\n\n\n<p>Their approach, outlined in full in a digital application note, introduces a practical workflow for multiplexing IHC and ISH in mouse brain tissue <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/in-situ-hybridization-ish\/rna-fish\/viewrna-assays.html\"><strong>using Invitrogen\u2122 ViewRNA\u2122 Tissue Assay Kits<\/strong><\/a> (fluorescence or colorimetric) alongside antibody-based IHC labeling.<\/p>\n\n\n\n<div class=\"wp-block-group is-layout-constrained wp-block-group-is-layout-constrained\">\n<div class=\"wp-block-group is-layout-constrained wp-block-group-is-layout-constrained\">\n<hr class=\"wp-block-separator has-text-color has-alpha-channel-opacity has-background is-style-dots\" style=\"background-color:#ee3134;color:#ee3134\" \/>\n<\/div>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:33.33%\">\n<figure class=\"wp-block-image size-full is-resized\"><a href=\"https:\/\/documents.thermofisher.com\/TFS-Assets\/BID\/Application-Notes\/multiplexing-immunohistochemistry-mrna-insitu-hybridization-app-note.pdf\" target=\"_blank\" rel=\" noreferrer noopener\"><img loading=\"lazy\" decoding=\"async\" width=\"551\" height=\"695\" src=\"https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/App-Note-Cover-1.jpg\" alt=\"\" class=\"wp-image-5048\" style=\"width:208px;height:auto\" srcset=\"https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/App-Note-Cover-1.jpg 551w, https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/App-Note-Cover-1-238x300.jpg 238w\" sizes=\"auto, (max-width: 551px) 100vw, 551px\" \/><\/a><\/figure>\n<\/div>\n\n\n\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:66.66%\">\n<h4 class=\"wp-block-heading has-text-color has-link-color wp-elements-d30e78b517ca41ee44afae1c57c09882\" id=\"h-application-note-multiplexing-ihc-and-mrna-ish\" style=\"color:#ee3134\">Application note: multiplexing IHC and mRNA ISH<div class=\"glfh_linkContainer\"><\/div><\/h4>\n\n\n\n<p>This note (.pdf) addresses the challenges of integrating spatial proteomics and transcriptomics with a neuroscience focus. Through a combination of RNA ISH and IHC techniques, these adapted protocols demonstrates multiplex spatial omics on mouse brain sections. <\/p>\n\n\n\n<div class=\"wp-block-buttons is-layout-flex wp-block-buttons-is-layout-flex\">\n<div class=\"wp-block-button\"><a class=\"wp-block-button__link has-background wp-element-button\" href=\"https:\/\/documents.thermofisher.com\/TFS-Assets\/BID\/Application-Notes\/multiplexing-immunohistochemistry-mrna-insitu-hybridization-app-note.pdf\" style=\"border-radius:0px;background-color:#ee3134\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Download app note<\/strong><\/a><\/div>\n<\/div>\n<\/div>\n<\/div>\n\n\n\n<hr class=\"wp-block-separator has-text-color has-alpha-channel-opacity has-background is-style-dots\" style=\"background-color:#ee3134;color:#ee3134\" \/>\n<\/div>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-approach\">Approach<\/h3>\n\n\n\n<p>To overcome ISH and IHC standard protocol incompatibilities, our experts implemented two critical modifications:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>RNase inhibition<\/strong> using recombinant ribonuclease inhibitors (<a href=\"https:\/\/www.thermofisher.com\/order\/catalog\/product\/10777019\" target=\"_blank\" rel=\"noreferrer noopener\">Invitrogen\u2122 RNaseOUT\u2122 recombinant ribonuclease inhibitor<\/a>) to preserve RNA during antibody incubation.<\/li>\n\n\n\n<li><strong>Antibody crosslinking<\/strong> after IHC labeling to protect protein signals from loss during ISH pretreatments.<\/li>\n<\/ul>\n\n\n\n<p>For RNA detection, they applied branched-DNA ISH probes (<strong><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/in-situ-hybridization-ish\/rna-fish\/viewrna-assays.html\" target=\"_blank\" rel=\"noreferrer noopener\">ViewRNA ISH kits<\/a>)<\/strong> capable of resolving up to four mRNA targets simultaneously, using either fluorescent readouts (<a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/antibodies\/antibody-labeling.html\"><strong>Invitrogen\u2122 Alexa F<\/strong><\/a><strong><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/antibodies\/antibody-labeling.html\" target=\"_blank\" rel=\"noreferrer noopener\">l<\/a><\/strong><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/antibodies\/antibody-labeling.html\"><strong>uor\u2122 dyes<\/strong><\/a>) or enzymatic colorimetric detection (Fast Red, Fast Blue, DAB).<\/p>\n\n\n\n<p>For protein detection, they used spectrally distinct antibodies, either pre-conjugated off-the-shelf or prepared in-house with the<a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/antibodies\/antibody-labeling.html\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Invitrogen\u2122 ReadyLabel\u2122 Antibody Labeling Kits<\/strong><\/a>. They also carefully designed panels to minimize spectral overlap and reduce autofluorescence, particularly in green emission channels.<\/p>\n\n\n\n<p>The team also prepared both cryopreserved and FFPE brain tissue, balancing the higher RNA integrity of cryosections against the lower RNase activity in FFPE samples. They then imaged samples across multiple platforms, ranging from widefield systems to a spectral imaging instrument that resolves up to nine fluorophores simultaneously.<\/p>\n\n\n\n<p>\ud83d\udc49 <em>Figure 1 shows why these workflow modifications are essential: with RNase inhibition, RNA and protein signals remain intact; without it, RNA signals nearly disappear despite strong antibody labeling.<\/em><\/p>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure data-wp-context=\"{&quot;imageId&quot;:&quot;6ab2bbc8cb6fa&quot;}\" data-wp-interactive=\"core\/image\" data-wp-key=\"6ab2bbc8cb6fa\" class=\"wp-block-image aligncenter size-large wp-lightbox-container\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"392\" data-wp-class--hide=\"state.isContentHidden\" data-wp-class--show=\"state.isContentVisible\" data-wp-init=\"callbacks.setButtonStyles\" data-wp-on--click=\"actions.showLightbox\" data-wp-on--load=\"callbacks.setButtonStyles\" data-wp-on-window--resize=\"callbacks.setButtonStyles\" src=\"http:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F1-Spatial-Blog-1024x392.jpg\" alt=\"Figure demonstrating the importance of using RNase inhibition in antibody labelling\" class=\"wp-image-5045\" srcset=\"https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F1-Spatial-Blog-1024x392.jpg 1024w, https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F1-Spatial-Blog-300x115.jpg 300w, https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F1-Spatial-Blog-768x294.jpg 768w, https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F1-Spatial-Blog.jpg 1127w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><button\n\t\t\tclass=\"lightbox-trigger\"\n\t\t\ttype=\"button\"\n\t\t\taria-haspopup=\"dialog\"\n\t\t\taria-label=\"Enlarge\"\n\t\t\tdata-wp-init=\"callbacks.initTriggerButton\"\n\t\t\tdata-wp-on--click=\"actions.showLightbox\"\n\t\t\tdata-wp-style--right=\"state.imageButtonRight\"\n\t\t\tdata-wp-style--top=\"state.imageButtonTop\"\n\t\t>\n\t\t\t<svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"12\" height=\"12\" fill=\"none\" viewBox=\"0 0 12 12\">\n\t\t\t\t<path fill=\"#fff\" d=\"M2 0a2 2 0 0 0-2 2v2h1.5V2a.5.5 0 0 1 .5-.5h2V0H2Zm2 10.5H2a.5.5 0 0 1-.5-.5V8H0v2a2 2 0 0 0 2 2h2v-1.5ZM8 12v-1.5h2a.5.5 0 0 0 .5-.5V8H12v2a2 2 0 0 1-2 2H8Zm2-12a2 2 0 0 1 2 2v2h-1.5V2a.5.5 0 0 0-.5-.5H8V0h2Z\" \/>\n\t\t\t<\/svg>\n\t\t<\/button><figcaption class=\"wp-element-caption\"><strong>Figure 1. <\/strong>\u00a0Comparison of the effect of antibody incubation \u00b1 RNaseOUT Recombinant Ribonuclease Inhibitor pre-treatment addition. (3a) Strong signal of GFAP and mRNA signal of Gad2 (yellow) and Ppib (red) with RNaseOUT pretreatment. (3b) Without RNaseOUT pretreatment, GFAP (green) antibody signal intensity is unaffected, but mRNA signal for both Gad2 (yellow) and Ppib (red) are greatly diminished. 40x images of the hippocampal region of a cryo mouse brain coronal section are gain and exposure matched. Images acquired on EVOS M7000 Imaging System.<\/figcaption><\/figure>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-results\">Results<\/h3>\n\n\n\n<p>The team\u2019s optimized protocol helped retain both RNA and protein signals in multiplex assays, successfully demonstrating that ISH and IHC can coexist in the same tissue section.<\/p>\n\n\n\n<p>The end result was high-content spatial analysis without specialized equipment:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>High-plex IHC:<\/strong> They generated an 8+1 antibody panel in mouse brain using iterative labeling with amplification reagents.<\/li>\n\n\n\n<li><strong>RNA detection:<\/strong> They simultaneously visualized four mRNA targets (Gad2, Ppib, Polr2a, Gapdh) with high sensitivity in both cryopreserved and FFPE tissue.<\/li>\n\n\n\n<li><strong>Colorimetric ISH:<\/strong> DAB substrates provided stable brightfield signals with faster development times, enabling archiving without loss of image quality.<\/li>\n\n\n\n<li><strong>Dual ISH + IHC:<\/strong> They preserved GFAP and HuC\/HuD protein signals while detecting Gad2 and Ppib RNA, revealing intricate neuronal patterns in hippocampal regions.<\/li>\n<\/ul>\n\n\n\n<p>\ud83d\udc49 <em>Figure 2 highlights the outcome: a multiplexed hippocampal section that maps both RNA and protein markers in situ, capturing the spatial complexity of neuronal populations.<\/em><\/p>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure data-wp-context=\"{&quot;imageId&quot;:&quot;6ab2bbc8cbd2b&quot;}\" data-wp-interactive=\"core\/image\" data-wp-key=\"6ab2bbc8cbd2b\" class=\"wp-block-image aligncenter size-full wp-lightbox-container\"><img loading=\"lazy\" decoding=\"async\" width=\"598\" height=\"323\" data-wp-class--hide=\"state.isContentHidden\" data-wp-class--show=\"state.isContentVisible\" data-wp-init=\"callbacks.setButtonStyles\" data-wp-on--click=\"actions.showLightbox\" data-wp-on--load=\"callbacks.setButtonStyles\" data-wp-on-window--resize=\"callbacks.setButtonStyles\" src=\"https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F2-Spatial-Blog.png\" alt=\"Stitched image of hippocampal mouse brain showing ISH + IHC multiplexing\" class=\"wp-image-5046\" srcset=\"https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F2-Spatial-Blog.png 598w, https:\/\/admin.acceleratingscience.com\/life-in-the-lab\/wp-content\/uploads\/sites\/10\/2025\/10\/F2-Spatial-Blog-300x162.png 300w\" sizes=\"auto, (max-width: 598px) 100vw, 598px\" \/><button\n\t\t\tclass=\"lightbox-trigger\"\n\t\t\ttype=\"button\"\n\t\t\taria-haspopup=\"dialog\"\n\t\t\taria-label=\"Enlarge\"\n\t\t\tdata-wp-init=\"callbacks.initTriggerButton\"\n\t\t\tdata-wp-on--click=\"actions.showLightbox\"\n\t\t\tdata-wp-style--right=\"state.imageButtonRight\"\n\t\t\tdata-wp-style--top=\"state.imageButtonTop\"\n\t\t>\n\t\t\t<svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"12\" height=\"12\" fill=\"none\" viewBox=\"0 0 12 12\">\n\t\t\t\t<path fill=\"#fff\" d=\"M2 0a2 2 0 0 0-2 2v2h1.5V2a.5.5 0 0 1 .5-.5h2V0H2Zm2 10.5H2a.5.5 0 0 1-.5-.5V8H0v2a2 2 0 0 0 2 2h2v-1.5ZM8 12v-1.5h2a.5.5 0 0 0 .5-.5V8H12v2a2 2 0 0 1-2 2H8Zm2-12a2 2 0 0 1 2 2v2h-1.5V2a.5.5 0 0 0-.5-.5H8V0h2Z\" \/>\n\t\t\t<\/svg>\n\t\t<\/button><figcaption class=\"wp-element-caption\"><strong>Figure 2a<\/strong>. Stitched image of hippocampal region of cryopreserved mouse brain showing IHC + ISH multiplexing of DAPI (blue), GFAP (green), Gad2 (yellow), Ppib (magenta), and HuC\/D (red) were acquired using a 20x objective on the EVOS S1000 Spatial Imaging System. <strong>Figure 2b.<\/strong> Panel showing the individual and combined channels of a small region of interest.\u00a0<\/figcaption><\/figure>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"h-spatial-biology-and-multi-omics-technologies\">Spatial biology and multi-omics technologies<\/h2>\n\n\n\n<p>While specialized equipment is not always necessary for advanced analysis in spatial biology, a strong collection of core multipurpose instruments can move the needle across the board.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"h-imaging-platforms\">Imaging platforms<\/h2>\n\n\n\n<p>Imaging platforms play a central role in multiplex workflows, since accurate signal detection depends on both sensitivity and spectral resolution. Systems capable of spectral unmixing and multi-channel acquisition allow researchers to visualize numerous RNA and protein targets simultaneously, while also offering flexibility for brightfield or fluorescence readouts depending on the assay design.<\/p>\n\n\n\n<p>Platforms range from versatile systems like the <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/evos-cell-imaging-systems\/models\/evos-m5000.html\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Invitrogen\u2122 EVOS\u2122 M5000<\/strong><\/a> to specialized instruments such as the <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/evos-cell-imaging-systems\/models\/tissue-imager-evos-s1000.html\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Invitrogen\u2122<\/strong> <strong>EVOS\u2122 S1000 Spatial Imaging System<\/strong><\/a>, which can simultaneously resolve nine fluorophores during a single acquisition with integrated spectral unmixing. The right imaging system can make or break complex spatial biology experiments.<\/p>\n\n\n\n<p><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/fluorescence-microscopy-and-immunofluorescence-if\/mounting-medium-antifades.html\" target=\"_blank\" rel=\"noreferrer noopener\">Mountants<\/a> like <a href=\"https:\/\/www.thermofisher.com\/order\/catalog\/product\/P38930\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>ProLong\u2122 RapidSet\u2122<\/strong><\/a>also play a subtle but critical role in spatial imaging. By preventing photobleaching and maintaining stable colorimetric deposits, they ensure that multiplexed ISH and IHC signals remain bright, balanced, and reliable for both immediate analysis and long-term archiving.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-rna-detection\">RNA detection<\/h3>\n\n\n\n<p>Signal amplification strategies become essential when pushing detection limits. ViewRNA assay techniques employ pairs of single-stranded DNA oligomers that hybridize to mRNA targets, forming a double-Z structure. This structure undergoes amplification through successive rounds of branched DNA oligomer hybridization under high stringency conditions, creating a substantially amplified complex with minimal background noise.<\/p>\n\n\n\n<p>The ViewRNA assay portfolio includes both fluorescence and colorimetric detection options, with the fluorescence kit enabling simultaneous visualization of up to four RNA targets using Alexa Fluor dyes (488, 546\/594, 647, and 750). Alternatively, the colorimetric kits utilize enzymatic signal amplification through alkaline phosphatase (with Fast Red or Fast Blue substrates) or horseradish peroxidase (with DAB solution).<\/p>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"h-protein-detection\">Protein detection<\/h3>\n\n\n\n<p>For protein detection, researchers can use either directly labeled antibodies or label unconjugated antibodies using the ReadyLabel Antibody Labeling Kit. The latter approach offers flexibility when directly labeled antibodies are unavailable for specific targets.<\/p>\n\n\n\n<p>Spatial amplification reagents like <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/immunofluorescence\/aluora-spatial-amplification.html\" target=\"_blank\" rel=\"noreferrer noopener\">Invitrogen\u2122 Aluora\u2122 Spatial Amplification Reagents<\/a> make it possible to detect many protein targets in the same tissue by boosting signal intensity and enabling iterative rounds of labeling. This approach not only enhances weak signals but also supports high-plex antibody panels, creating a strong foundation for combining protein and RNA detection in a single workflow.<\/p>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"h-key-takeaways\">Key takeaways<\/h2>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Dual ISH\u2013IHC workflows can reliably preserve both RNA and protein signals.<\/strong> Strategic use of RNase inhibition and antibody crosslinking overcomes the incompatibilities that typically cause signal loss.<\/li>\n\n\n\n<li><strong>Multiplexing can deliver deeper biological insight.<\/strong> High-plex antibody panels combined with multi-target RNA assays reveal cell states, heterogeneity, and tissue interactions with greater clarity.<\/li>\n\n\n\n<li><strong>This workflow applies well beyond neuroscience.<\/strong> Researchers studying cancer, immunology, and aging can adapt the same approach to interrogate diverse tissue contexts.<\/li>\n\n\n\n<li><strong>Advanced spatial multi-omics does not require highly specialized systems.<\/strong> With optimized reagents and widely available imaging platforms, complex workflows become accessible to many labs.<\/li>\n\n\n\n<li><strong>Small protocol adjustments can enable big experimental gains.<\/strong> The application note demonstrates how fine-tuning established methods transforms spatial biology from incremental to integrative discovery.<\/li>\n<\/ul>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"h-more-spatial-biology-and-multi-omics-resources\">More spatial biology and multi-omics resources<\/h2>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Application Note:<\/strong> <a href=\"https:\/\/documents.thermofisher.com\/TFS-Assets\/BID\/Application-Notes\/multiplexing-immunohistochemistry-mrna-insitu-hybridization-app-note.pdf\" target=\"_blank\" rel=\"noreferrer noopener\">Multiplexing IHC and mRNA ISH with a neuroscience focus<\/a><\/li>\n\n\n\n<li><strong>Explore: <\/strong><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/c\/b\/enhanced-imaging-optimized-solutions.html?icid=fl-bid-imagingpairs\" target=\"_blank\" rel=\"noreferrer noopener\">Enhanced imaging with optimized solutions<\/a><\/li>\n\n\n\n<li><strong>Learn:<\/strong> <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cell-analysis-learning-center\/spatial-biology.html\" target=\"_blank\" rel=\"noreferrer noopener\">Spatial biology resource center<\/a><\/li>\n\n\n\n<li><strong>Learn: <\/strong>Immunohistochemistry (IHC)<ul><li><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/protein-biology\/protein-biology-learning-center\/protein-biology-resource-library\/pierce-protein-methods\/overview-immunohistochemistry.html\">Overview of IHC<\/a><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/antibodies\/primary-antibodies\/antibodies-applications\/antibodies-immunohistochemistry.html\" target=\"_blank\" rel=\"noreferrer noopener\">Antibodies for IHC<\/a><\/li><\/ul>\n<ul class=\"wp-block-list\">\n<li><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/ihc.html\" target=\"_blank\" rel=\"noreferrer noopener\">5 steps for great IHC images<\/a><\/li>\n<\/ul>\n<\/li>\n\n\n\n<li><strong>Learn: <\/strong>In situ hybridization (ISH)\n<ul class=\"wp-block-list\">\n<li><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/in-situ-hybridization-ish\/rna-fish.html\" target=\"_blank\" rel=\"noreferrer noopener\">RNA FISH assays<\/a><\/li>\n<\/ul>\n<\/li>\n\n\n\n<li><strong>Blog: <\/strong><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/brands\/thermo-scientific\/molecular-biology\/molecular-biology-learning-center\/molecular-biology-resource-library\/spotlight-articles\/supporting-multi-omics-approaches.html\" target=\"_blank\" rel=\"noreferrer noopener\">A commonality between various omes in multi-omics approaches<\/a><\/li>\n<\/ul>\n\n\n\n<div style=\"height:15px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<div class=\"wp-block-buttons is-content-justification-center is-layout-flex wp-container-core-buttons-is-layout-16018d1d wp-block-buttons-is-layout-flex\">\n<div class=\"wp-block-button has-custom-width wp-block-button__width-75 is-style-fill\"><a class=\"wp-block-button__link has-white-color has-text-color has-background has-link-color has-medium-font-size has-text-align-center has-custom-font-size wp-element-button\" href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/in-situ-hybridization-ish\/rna-fish\/viewrna-assays.html\" style=\"border-radius:0px;background-color:#ee3134\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Explore Invitrogen ViewRNA ISH assays<\/strong><\/a><\/div>\n<\/div>\n\n\n\n<div style=\"height:25px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p><em>##<\/em>&nbsp;<\/p>\n\n\n\n<p><em>For Research Use Only. Not for use in diagnostic procedures.&nbsp;<\/em>&nbsp;&nbsp;<\/p>\n\n\n\n<p>\u00a9 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Spatial omics biology is reshaping how researchers study complex tissues by preserving the molecular context that traditional methods often lose. For scientists pushing toward true spatial multi-omics, integrating immunohistochemistry (IHC) with in situ hybridization (ISH) represents both an opportunity and a technical challenge. This blog explores a recent application note that demonstrates how careful protocol<\/p>\n","protected":false},"author":1670,"featured_media":5043,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[976,4],"tags":[],"division":[],"class_list":{"0":"post-5042","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-connect-to-science","8":"category-general","9":"entry"},"_selected_authors":[744],"_selected_reviewers":[],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Spatial Omics x Neuroscience: Dual ISH-IHC Brain Mapping<\/title>\n<meta name=\"description\" content=\"Through a combination of RNA ISH and IHC techniques, these adapted protocols demonstrate multiplex spatial omics on mouse brain sections.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.thermofisher.com\/blog\/life-in-the-lab\/spatial-omics-dual-ish-ihc-brain-mapping\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Spatial Omics Meets Neuroscience: Dual ISH-IHC Brain Mapping Case Study\" \/>\n<meta property=\"og:description\" content=\"Through a combination of RNA ISH and IHC techniques, these adapted protocols 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