High resolution platinum-carbon replication of freeze-dried basement membrane

High angle platinum/carbon (Pt/C) replication has proved to be a valuable tool in analyzing basement membrane structure in human amnion, bovine lens capsule, and the Engelbreth-Holm-Swarm (EHS) tumor. High resolution replicas for transmission electron microscopy (TEM) have been achieved by depositing 1.0 +/- 0.1 nm thick Pt/C films backed with rotary deposited 12.5 +/- 2.5 nm thick carbon films. The basement membrane collagen IV network was observed to consist of fine branching filaments containing globular domains intrinsic to the filaments. A second quasi-regular network is formed by laminin. Unidirectional 45 degree angle Pt/C replication was used for most of this work. The merits and deficiencies of unidirectional vertical replication (80 degree angle), unidirectional 45 degree angle, and 20 degrees low angle rotary replication are discussed. Vertical replication produces the highest resolution replicas and has the potential for revealing the overall pattern of basement membrane structural assembly if basement membrane preparations freeze-dried in low salt can faithfully maintain their in vivo structure.