Localization of heparan sulfate proteoglycan in basement membrane by side chain staining with cuprolinic blue as compared with core protein labeling with immunogold.

Abstract

We localized heparan sulfate proteoglycan (HSPG) in the basement membranes of ciliary epithelium and plantar epidermis, using Cuprolinic blue to stain its side chains and an immunogold procedure to detect its core protein. In accord with most of the literature, staining with Cuprolinic blue in glutaraldehyde fixative yielded three to five times as many reaction products along the two surfaces than along the center of the lamina densa, whereas immunogold labeling for the core protein after formaldehyde fixation yielded about twice as many gold particles over the center than along the surfaces of the lamina densa. It therefore appeared that HSPG side chains predominated outside, and the core protein within, the lamina densa. To find out whether the discrepancy was true or was an artifact caused by differences in processing, we attempted to combine the two approaches on the same material. This was found possible when Cuprolinic blue was used in formaldehyde fixative, embedding was in LR White, and immunogold labeling was performed on thin sections as usual. Under these conditions, both Cuprolinic blue reaction products and immunogold particles predominated over the lamina densa in the two basement membranes under study. Moreover, evidence was present that reaction products and immunogold particles either overlapped each other or were closely associated. The lens capsule (a thick basement membrane) also showed their co-localization. The discrepancy initially observed between side chains and core protein location was attributed to differences in processing, since Cuprolinic blue staining had been carried out in the course of glutaraldehyde fixation whereas immunogold labeling was done after formaldehyde fixation. The results lead to two conclusions. First, processing differences may alter the localization of HSPG and possibly other proteoglycans. Second, both HSPG side chains and core protein are localized in the same sites within basement membrane.