THE BASEMENT-MEMBRANES OF CRYOFIXED OR ALDEHYDE-FIXED, FREEZE-SUBSTITUTED TISSUES ARE COMPOSED OF A LAMINA DENSA AND DO NOT CONTAIN A LAMINA-LUCIDA

When tissues are processed for electron microscopy by conventional met hods, such as glutaraldehyde fixation followed by rapid dehydration in acetone, basement membranes show two main layers: the electron-lucent ''lamina lucida'' (or rara) and the electron-dense ''lamina densa''. In an attempt to determine whether this subdivision is real or artefac tual, two approaches have been used. Firstly, rat and mouse seminifero us tubules, mouse epididymis and associated tissues, and anterior part s of mouse eyes were subjected to cryofixation by instant freezing fol lowed by freeze substitution in a -80-degrees-C solution of osmium tet roxide in dry acetone, which was gradually warmed to room temperature over a 3-day period. The results indicate that, in areas devoid of ice crystals, basement membranes consist of a lamina densa in direct cont act with the plasmalemma of the associated cells without an intervenin g lamina lucida. Secondly, a series of tissues from mice perfused with 3% glutaraldehyde were cryoprotected in 30% glycerol, frozen in Freon 22 and subjected to a 3-day freeze substitution in osmium tetroxide-a cetone as above. Under these conditions, no lamina lucida accompanies the lamina densa in the basement membranes of the majority of tissues, including kidney, thyroid gland, smooth and skeletal muscle, ciliary body, seminiferous tubules, epididymis and capillary endothelium. Thus , even though these tissues have been fixed in glutaraldehyde, no lami na lucida appears when they are slowly dehydrated by freeze substituti on. It is concluded that the occurrence of this lamina in conventional ly processed tissues is not due to fixation but to the rapid dehydrati on. However, in this series of experiments, the basement membranes of trachea and plantar epidermis include a lamina lucida along their enti re length, while those of esophagus and vas deferens may or may not in clude a lamina lucida. To find out if the lamina lucida appearing unde r these conditions is a real structure or an artefact, the trachea and epidermis were fixed in paraformaldehyde and slowly dehydrated by fre eze substitution. Under these conditions, no lamina lucida was found. Since this result is the same as observed in other tissues by the prev ious approaches, it is proposed that the lamina lucida is an artefact in these as in the other investigated basement membranes. Thus, baseme nt membranes are simply composed of a lamina densa that closely follow s the plasmalemma of the associated cells. At high magnification, the lamina densa consists of a tridimensional network of cords, while the plasmalemma is covered by a glycocalyx; close contact is observed betw een cords and glycocalyx and is interpreted by assuming that the lamin in present in the cords binds to laminin receptors in the glycocalyx.