Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections
June 25th, 2021
8.3K views
This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.
Overview
This study presents a protocol for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) on fresh frozen and fixed mouse brain sections. The technique allows for the spatial visualization of RNA and protein targets simultaneously, aiding in the investigation of neuronal populations.
Key Study Components
Area of Science
- Neuroscience
- Molecular biology
- Immunohistochemistry
Background
- The study investigates the spatial organization of mRNA and proteins in heterogeneous neuronal populations.
- Combining FISH and IHC enables multiplex labeling in brain tissue sections.
- It aims to improve the understanding of gene expression and protein localization within the brain.
- The protocol is applicable to both fresh-frozen and paraformaldehyde-fixed tissues.
Purpose of Study
- To demonstrate a reliable and flexible method of simultaneous RNA and protein detection.
- To enhance the investigation of mRNA distribution and protein localization in neuronal cells.
- To validate the quality of immunohistochemical labeling when combined with FISH.
Methods Used
- Fresh-frozen and paraformaldehyde-fixed brain sections were used as the main platform.
- The biological model involved mouse brain tissue, focusing on RNA and protein signals in neurons.
- The method includes critical steps like sectioning, fixation, hybridization, and signal amplification.
- Detailed incubation and washing protocols were followed to ensure clear results.
Main Results
- Positive control probes confirmed RNA integrity, while varying degrees of signal clarity in proteins were observed.
- The study demonstrated the ability to differentiate between cytoplasmic and nuclear signals in neurons.
- Indications of distinct neuronal populations and their RNA/protein expressions were reported.
- The combination of FISH and IHC proved reliable for visualizing multiple targets within the same sections.
Conclusions
- The protocol enables advanced exploration of neuron diversity and interaction within brain tissue.
- This study provides a framework for future investigations into neuronal mechanisms and potential disease models.
- The method enriches our understanding of molecular and cellular dynamics in neuroscience.
Frequently Asked Questions
What are the advantages of using this protocol?
This protocol allows for the simultaneous visualization of RNA and protein targets, which enhances understanding of cellular functions in the brain.
How is the biological model implemented?
The model uses mouse brain tissue sections processed through specific sectioning, fixation, and labeling protocols to investigate neuronal populations.
What types of data can be obtained from this method?
The method provides qualitative and quantitative data regarding mRNA distribution and protein expression in specific neuronal populations.
How can this method be applied in other studies?
Researchers can adapt this technique for various studies involving gene expression and protein localization in different tissue types and species.
What are key considerations when using this protocol?
It is crucial to ensure tissue integrity and adherence to the outlined steps to avoid disruptions in signal detection.
Are there limitations to this approach?
The reliance on specific fixation methods may limit the applicability to certain proteins or RNA, necessitating adjustments based on experimental needs.
Executive Industry Relevance
Combining multiplex FISH with fluorescence IHC enables biopharma R&D to resolve spatial co-expression of RNA and protein targets within heterogeneous neuronal populations, supporting target validation and mechanistic de-risking in neuroscience drug discovery. This approach enhances predictive confidence by linking molecular signatures to cellular phenotypes, informing go/no-go decisions in early discovery. The method’s applicability to fresh-frozen and fixed tissues provides workflow flexibility for target interrogation across preclinical models.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by mapping low-abundance mRNAs (e.g., galanin receptor 1) and high-abundance transcripts within immunohistochemically defined brain nuclei.
- Operational Value: Supports biological de-risking through concurrent RNA-protein labeling, clarifying target engagement and pathway modulation in neuronal circuits.
- Predictive Value: Facilitates portfolio triage by distinguishing neuronal subpopulations based on neurochemical signatures, reducing ambiguity in target selection.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream screening by confirming RNA integrity via positive control probes (ubiquitin C, peptidylprolyl isomerase B, RNA polymerase IIA).
- Operational Value: Enables assay standardization through optimized protease incubation and hybridization conditions, improving reproducibility across fresh-frozen and fixed tissue preparations.
- Scalability Value: Supports platform reuse for multiplex RNA-protein detection, aiding reliable compound evaluation in phenotypic screening campaigns.
Translational & Preclinical Research
- Scientific Value: Enhances translational biomarker alignment by visualizing spatial organization of mRNA and protein expression in disease-relevant brainstem nuclei (e.g., NTS).
- Operational Value: Ensures continuity from discovery through preclinical validation by maintaining IHC quality post-FISH, particularly for membrane-bound targets.
- Risk-Adjusted Advancement: Informs decision-making by revealing lack of colocalization between GlyT2 mRNA-positive and PHOX2B mRNA-positive neurons, refining mechanistic models.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from target identification through lead optimization, enabling spatial validation of molecular targets in neuronal populations prior to phenotypic screening.
- Discovery Biology: Supports hypothesis testing and pathway clarification by resolving RNA distribution within immunohistochemically identified neurons, reducing mechanistic ambiguity.
- Screening: Delivers assay readiness through standardized FISH-IHC workflows, ensuring reproducible spatial readouts for compound response evaluation.
- Analytics: Generates quantitative spatial data via fluorescence signal detection, enabling comparison of RNA and protein expression levels across experimental conditions.
- Translational Research: Connects to preclinical continuity by validating target expression in fixed tissue preparations, supporting biomarker-aligned disease modeling.
- Enterprise Reuse: Functions as a reusable spatial profiling capability across neuroscience discovery programs, maximizing infrastructure investment.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence through direct visualization of RNA-protein co-expression, reducing false positives in target validation.
- Operational Value: Enhances standardization and reproducibility via optimized protease and hybridization steps, minimizing variability in multiplex labeling.
- Strategic Value: Improves capital efficiency by enabling early de-risking of targets through subcellular resolution, reducing late-stage attrition due to biological mismatch.
- Portfolio Impact: Supports risk-adjusted prioritization by identifying neuronal subpopulations with distinct RNA-protein profiles, informing indication selection.
Implementation Considerations
- Requires expertise in fluorescence microscopy, RNA probe handling, and antibody optimization to manage signal specificity.
- Dependent on RNase-free environments, cryostat or microtome sectioning tools, and fluorescence imaging systems with appropriate filter sets.
- Necessitates cross-team standardization of protease incubation times and antibody validation protocols to ensure consistent IHC quality post-FISH.
- Requires adaptation based on protein subcellular localization, as cytoplasmic targets may need extensive troubleshooting while membrane-bound proteins show retained specificity.
- Limited by tissue handling challenges in fixed-frozen preparations, though IHC quality is superior when optimized with RNAscope.
Biopharma FAQs
Why does protease treatment in FISH affect IHC specificity in multiplex labeling?
Protease treatment facilitates FISH probe penetration by cleaving peptide bonds but can digest cytoplasmic proteins targeted by subsequent IHC, reducing labeling specificity. Membrane-bound proteins retain IHC quality post-protease treatment, while cytoplasmic targets like tyrosine hydroxylase show flocculent, nonspecific signal. Optimization requires balancing FISH efficiency with antigen preservation based on target localization.
How does independent variable isolation support target validation in neuroscience discovery?
Isolating RNA as an independent variable via RNAscope FISH enables unambiguous attribution of signal changes to transcriptional regulation, independent of protein-level confounders. This approach was used to distinguish GalR1 mRNA expression patterns in the NTS from protein markers like PHOX2B and tyrosine hydroxylase. Independent variable control strengthens causal inference in target engagement studies.
What quantitative dependent variable measurements enable hit-to-lead progression in screening campaigns?
Fluorescence signal intensity and puncta count from RNAscope FISH serve as quantitative dependent variables for measuring mRNA expression levels in single cells. These metrics were used to detect low-abundance GalR1 mRNA and high-abundance GlyT2 mRNA in brainstem nuclei. Quantitative readouts allow rank-ordering of compounds based on target modulation potency.
Why are replication requirements critical for cross-functional collaboration in target validation?
Replication across fresh-frozen and fixed tissue preparations ensures that observed RNA-protein co-expression patterns are robust and not artifacts of a single processing method. The study replicated GlyT2 and PHOX2B mRNA localization in both tissue types, confirming consistent ventral NTS positioning. Reproducibility builds confidence across biology, chemistry, and translational teams advancing shared targets.
What statistical analysis capabilities are required before implementing multiplex FISH-IHC in lead identification?
Capabilities for colocalization analysis and signal thresholding are needed to quantify overlap between RNA and protein signals across neuronal populations. The study assessed lack of colocalization between GlyT2 mRNA-positive and PHOX2B mRNA-positive neurons using spatial fluorescence data. Statistical rigor ensures objective interpretation of multiplex imaging data for go/no-go decisions.
