Cryofixation of basement membranes followed by freeze substitution or freeze drying demonstrates that they are composed of a tridimensional network of irregular cords

Abstract

Since conventional chemical fixation may extract tissue components and thus alter structural organization, cryofixation was used to reexamine the ultrastructure of three thick basement membranes: lens capsule, Reichert's membrane, and Engelbreth-Holm-Swarm (EHS) tumor matrix, and two thin basement membranes, those of epididymis and seminiferous tubules. Cryofixation was achieved by slam freezing followed by either freeze substitution in dry acetone containing 1% osmium tetroxide and 0.05% uranyl acetate or freeze drying in a molecular distillation dryer. The results by both procedures demonstrate that thick basement membranes and the lamina densa of thin basement membranes are composed of a network of anastomosing strands referred to as cords. The cords vary in density and distinctiveness, but their thickness averages 3 to 5 nm in every tissue examined. The spaces separating the cords vary within wide limits, but their mean diameter is ∼15 nm in every case. Two other common features are (1) the presence within the network of a few 1.5–3.0-nm-thick filaments and (2) 4.5-nm-wide sets of parallel lines referred to as double tracks. When these results are compared with those previously described after conventional fixation, no significant difference is observed in either the cord network or the associated filaments and “double tracks.” However, in the thin basement membranes processed by cryofixation, the lamina densa is in direct contact with epithelial cells, whereas, after conventional fixation, the lamina densa is separated from the epithelial cells by a pale layer referred to as lamina lucida or lamina rara. Immunogold labeling of three basement membranes after cryofixation and freeze substitution in acetone containing 0.3% glutaraldehyde yields strong reactions for laminin, type IV collagen, and heparan sulfate proteoglycan. Comparison with previous results indicates that conventional formaldehyde fixation adequately preserves laminin and type IV collagen but causes the loss of some proteoglycan. It is concluded that, except for this loss and the absence of lamina lucida in cryofixed thin basement membranes, the morphological and antigenic features obtained after cryofixation are similar to those observed in the past after conventional fixation.