Related Experiment Video
Updated: Jul 15, 2026
Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections
Published on: June 25, 2021
Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified
Edith M Schneider Gasser1, Carolin J Straub, Patrizia Panzanelli
1Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Insights
New methods allow better visualization of synaptic proteins in the brain. These techniques overcome fixation issues, enabling clearer studies of inhibitory synapses and their function.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Synaptic molecular organization is key to brain function and plasticity.
- Immunofluorescence is crucial for morphological studies but limited by epitope masking post-fixation.
- Simultaneous visualization of pre- and postsynaptic proteins is challenging due to differential fixation requirements.
Purpose of the Study:
- To present novel protocols for improved synaptic protein detection.
- To overcome limitations of traditional perfusion fixation methods.
- To enable simultaneous visualization of inhibitory synaptic components.
Main Methods:
- Developed three alternative tissue fixation protocols.
- Utilized brief fixation post-vital function interruption.
- Employed specific neuronal markers and confocal laser scanning microscopy.
Main Results:
- Preserved morphology and antigenicity using brief fixation.
- Achieved selective detection of gamma-aminobutyric acid A (GABA(A)) receptors and gephyrin.
- Enabled visualization of inhibitory presynaptic terminals and postsynaptic proteins.
Conclusions:
- Novel fixation protocols enhance synaptic protein analysis in situ.
- These methods facilitate correlative studies of synapse structure and function, potentially with electrophysiology.
- The protocols offer a valuable alternative for studying inhibitory synapse organization.
Abstract:
Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of gamma-aminobutyric acid A (GABA(A)) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2-3 consecutive days for completion.
