Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons.
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.
Schneider Gasser
Edith M
EM
Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Straub
Carolin J
CJ
Panzanelli
Patrizia
P
Weinmann
Oliver
O
Sassoè-Pognetto
Marco
M
Fritschy
Jean-Marc
JM
D000818:Animals; D001923:Brain Chemistry; D005455:Fluorescent Antibody Technique; D051379:Mice; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000737:chemistry*; D051381:Rats; D013569:Synapses / Q000737:chemistry*
D009419:Nerve Tissue Proteins