Electron microscopic immunocytochemistry of interstitial retinol-binding protein in vertebrate retinas

Interstitial retinol binding protein (IRBP) is a soluble glycoprotein found in the interphotoreceptor matrix (IPM) and implicated in shuttling retinol between retina and pigment epithelium (PE) cells. The authors have studied the distribution of IRBP by EM immunocytochemistry. Thin sections of Lowicryl K4M embedded R. pipiens, X. laevis, bovine and human retinas were labeled sequentially with affinity purified rabbit antibovine IRBP, biotinyl-sheep antirabbit F(Ab')2, and avidin-ferritin, or with avidin and biotinyl-ferritin. Antigen was in the interphotoreceptor space and intercalated into the narrow spaces between PE cell microvilli. IRBP penetration between PE cells was delimited abruptly by the PE junctional complexes. IRBP was also observed in small vacuoles in the apical cytoplasm of PE cells and in PE cell phagosomes that contained IRBP surrounding ingested rod tips. IPM was heavily but inhomogeneously labeled. Antigen was usually deposited along the ROS and COS plasma membrane in a confluent layer, but sometimes it was distributed in large (ca. 0.2-micron thick) clumps. In bovine and human retinas, the connecting cilium was ensheathed by antigen at high density but an unlabeled halo surrounded its plasma membrane. The apical plasma membrane of the inner segment aligned along the connecting cilium was also densely coated by antigen. In both frog retinas, the ridges of the periciliary ridge complex (PRC) were coated with antigen. In none of the four species examined was Golgi labeling present. In bovine retinas, labeled vacuoles (granules) in the myoid region were found in very low numbers (15 vacuoles in 358 rod cells). Amphibian retinas also contained only small numbers of myoid vacuoles labeled by anti-IRBP. Absence of antibody binding to intracellular sites of synthesis in any of the cells that abut the interphotoreceptor matrix suggests that the antigen may be masked prior to its release from the synthetic cell(s) or that its level is below limits of detection.