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Sequential Indirect Dual Immunohistochemistry with Primary Rabbit Antibodies on Cochlear Sections Using an
Maria Luque1, Rudolf Glueckert1,2
1Department of Otorhinolaryngology, Medical University of Innsbruck, Innsbruck, Austria.
Insights
This study details a new immunohistochemical (IHC) protocol for visualizing multiple proteins in mouse inner ear tissue. The method uses two rabbit antibodies for simultaneous fluorescence or colorimetric detection, enabling protein interaction studies.
Area of Science:
- Neuroscience
- Cell Biology
- Histology
Background:
- Advanced immunohistochemical (IHC) techniques are crucial for in situ visualization of multiple molecules, aiding the study of protein interactions at the subcellular level.
- Simultaneous staining in tissue sections can indicate protein proximity, but protocols are often limited by the availability of primary antibodies from different host species.
- Studying protein interactions within the inner ear is vital for understanding cochlear neuron function.
Purpose of the Study:
- To present a detailed protocol for double immunohistochemical staining in mouse inner ear tissue.
- To enable simultaneous visualization of transmembrane ion channel proteins in cochlear neurons.
- To overcome limitations of antibody host species in indirect IHC.
Main Methods:
- Developed a protocol using two primary rabbit antibodies against cochlear neuron transmembrane ion channel proteins.
- Applied sequential single IHC stainings for fluorescence (confocal) and dual multiplex colorimetric visualization.
- Included a heat-denaturation step between stainings, antibody specificity testing, and tissue controls.
- Detailed tissue extraction, fixation, cryoembedding, and preparation for automated and manual immunostaining, including antigen retrieval.
Main Results:
- Successfully demonstrated a method for double IHC staining using antibodies from the same host species (rabbit).
- Validated antibody specificity through peptide preadsorption and confirmed reliability with positive and negative tissue controls.
- Established a comprehensive protocol applicable to both manual and automated immunostaining platforms.
Conclusions:
- The presented protocol facilitates double IHC staining in mouse inner ear tissue using antibodies from the same host species.
- This approach expands possibilities for detecting multiple targets in single tissue sections, utilizing widely available resources.
- The method is valuable for studying protein proximity and interactions in cochlear neurons.
Abstract:
Advanced immunohistochemical (IHC) protocols aim to visualize different molecules in situ simultaneously. These techniques are of utmost importance as a first step in studying possible interactions of proteins at the subcellular level. Colocalized stains in tissue sections indicate proximity of two proteins of interest. Frequently, double staining protocols are restricted by the lack of primary antibodies generated in different animal species for indirect IHC visualization. Here, we present a detailed protocol for mouse inner ear tissue using two different primary rabbit antibodies directed against transmembrane ion channel proteins of cochlear neurons. The two antibodies are combined for fluorescence (confocal) as well as dual multiplex colorimetric visualization in two sequential single IHC stainings. A heat-denaturation step is performed in between. Primary antibody specificity is tested by preadsorption with the immunogenic peptide, and positive and negative tissue controls are performed to confirm the reliability of the antibody detection. We describe the whole procedure in detail beginning with tissue extraction of the mouse inner ear and continuing with chemical fixation, cryoembedding, and preparation for manual and fully automated immunostaining, including steps for heat-induced antigen retrieval. The potential to use antibodies from the same host species for single and double IHC staining opens up multiple possibilities for detecting different targets in the same tissue section using resources and materials that are widely available. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue preparation, cryoembedding, and sectioning Basic Protocol 2: Double colorimetric immunostaining with an automatic immunostainer Basic Protocol 3: Double manual fluorometric immunostaining with fluorescence.
