title: Improved immunohistochemical detection of postsynaptically located PSD-95/SAP90 protein family by protease section pretreatment: a study in the adult mouse brain.
abstract: Postsynaptic density (PSD)-95, SAP102, and Chapsyn-110 are members of the PSD-95/SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD-95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits.
authors.affiliation: Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Fukaya
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D000076122:Disks Large Homolog 4 Protein; D005455:Fluorescent Antibody Technique; D051528:Guanylate Kinases; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051379:Mice / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019581:Neuropil / Q000378:metabolism; D010434:Pepsin A / Q000494:pharmacology*; D000074407:SAP90-PSD95 Associated Proteins; D013569:Synapses / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D000074407:SAP90-PSD95 Associated Proteins; C033652:postsynaptic density proteins; C501445:Dlg2 protein, mouse; D051528:Guanylate Kinases; D010434:Pepsin A