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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.
Current Protocols
|September 8, 2021
Summary
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.
