A Highly Sensitive Immunofluorescence Procedure for Analyzing the Subcellular Distribution of GABA A Receptor Subunits in the Human Brain

• Published in: The journal of histochemistry and cytochemistry Vol. 46; no. 10; pp. 1129 - 1139
• Main Authors: Loup, Fabienne, Weinmann, Oliver, Yonekawa, Yasuhiro, Aguzzi, Adriano, Wieser, Heinz-Gregor, Fritschy, Jean-Marc
• Format: Journal Article
• Language: English
• Published: 01.10.1998
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1177/002215549804601005

We designed a protocol to improve the immunohistochemical analysis of human brain structures, which overcomes the limited detection sensitivity, high background, and intense autofluorescence commonly associated with human tissue. This procedure was evaluated by using antibodies against major GABA A receptor subunits (α 1 , α 2 , α 3 , γ 2 ) in autopsy and surgical specimens. Tissue blocks were briefly fixed by immersion and pretreated with microwave irradiation in sodium citrate buffer. Immunoperoxidase staining revealed a marked enhancement of cell surface immunoreactivity and reduction of background in microwave- irradiated tissue, irrespective of its origin. For confocal laser scanning microscopy, immunofluorescence staining was optimized with the tyramide signal amplification (TSA) technique. This procedure not only dramatically increased the sensitivity for antigen detection but also totally suppressed autofluorescence, thus revealing the cellular and subcellular distribution of GABA A receptor subunits. A distinct neuron-specific expression pattern of the α-subunit variants was observed in cerebral cortex and hippocampal formation, along with widespread expression of the γ 2 -subunit. Of particular interest was the prominent α 2 - and α 3 -subunit staining on the initial axon segment of pyramidal neurons. This protocol represents a major improvement for high-resolution studies of human brain tissue aimed at investigating morphological alterations underlying neurological diseases.