Localization of heparan sulfate proteoglycan in basement membranes.

Abstract

In a recent issue of the Journal (1). we reexamined the localization of heparan sulfate proteoglycan (HSPG) in basement membranes in the hope of settling a controversy opposing investigators who used cationic probes and localized HSPG anionic groups mainly to the lamina lucida (3-5,9) and other researchers who used antibodies to HSPG core protein and localized it mainly to the lamina densa (6,7). We attributed the discrepancy to the use of glutaraldehyde fixation by cationic probe users and formaldehyde fixation by antibody users. When the tests for the anionic groups and core protein of HSPG were combined on the same sections, both were localized mainly in the lamina densa. However, the combined tests could be carried out after formaldehyde fixation but not after glutaraldehyde fixation. We therefore could not safely conclude that HSPG was localized in the lamina densa. The problem was recently settled when various tissues processed either by cryofixation and freeze substitution or by glutaraldehyde fixation, followed by freezing in Freon and freeze-substitution, showed nine different basement membranes composed of a lamina densa without lamina lucida, as illustrated in the mouse glomerular basement membrane (Figure 1). It wasconcluded, in accord with previous findings (2.8). that the lamina lucida was an artifact, at least in the nine tissues investigated. Moreover gold immunolabeling of basement membranes processed in this manner with antibodies against HSPG core protein localized this substance throughout the basement membrane (Figure 1). Counts of the number of gold particles per 0.1 pm on nine micrographs gave 0.83 2 0.33 in the proximal third (endothelial side), 1.18 * 0.33 in the central third (middle), and 0.79 * 0.25 in the distal third (visceral side) of this basement membrane. Such density was on the same order of magnitude as that reported in the basement membrane of the ciliary epithelium (1). Briefly, the controversy