A HIGHLY SENSITIVE IMMUNOFLUORESCENCE PROCEDURE FOR ANALYZING THE SUBCELLULAR-DISTRIBUTION OF GABA(A) RECEPTOR SUBUNITS IN THE HUMAN BRAIN

We designed a protocol to improve the immunohistochemical analysis of human brain structures, which overcomes the limited detection sensitiv ity, high background, and intense autofluorescence commonly associated with human tissue. This procedure was evaluated by using antibodies a gainst major GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3), gamma(2)) in autopsy and surgical specimens. Tissue blocks were briefl y fixed by immersion and pretreated with microwave irradiation in sodi um citrate buffer. Immunoperoxidase staining revealed a marked enhance ment of cell surface immunoreactivity and reduction of background in m icrowave-irradiated tissue, irrespective of its origin. For confocal l aser scanning microscopy, immunofluorescence staining was optimized wi th the tyramide signal amplification (TSA) technique. This procedure n ot only dramatically increased the sensitivity for antigen detection b ut also totally suppressed autofluorescence, thus revealing the cellul ar and subcellular distribution of GABAA receptor subunits. A distinct neuron-specific expression pattern of the alpha-subunit variants was observed in cerebral cortex and hippocampal formation, along with wide spread expression of the gamma(2)-subunit. Of particular interest was the prominent alpha(2)- and alpha(3)-subunit staining on the initial a xon segment of pyramidal neurons. This protocol represents a major imp rovement for high-resolution studies of human brain tissue aimed at in vestigating morphological alterations underlying neurological diseases .