The use of inert dehydration and glycol methacrylate embedding for immunogold localization of glomerular basement membrane components.
A protocol is described for the preparation of human pathology specimens without fixation, in order to perform immunocytochemistry at an ultrastructural level. Using the technique in conjunction with immunogold labeling, the basal lamina components type IV collagen, laminin, and heparan sulfate have been demonstrated in glomerular capillary loops in stored frozen human renal tissue. Tissue was thawed and immediately dehydrated with the inert cryoprotectant ethanediol (inert dehydration) followed by embedding in low-acid glycol methacrylate polymerized using the accelerator n,n-dimethylaniline. Tissue processed in this way retained superior antigenic activity when compared with tissue reprocessed from wax blocks and embedded in low-acid glycol methacrylate. Inert dehydration is a technique useful for the localization of processing sensitive epitopes in routine fresh or frozen archival pathologic material. Furthermore, high probe densities can be achieved without recourse to etching or enzyme treatments.
Stirling
J W
JW
Department of Pathology, Flinders Medical Centre, Bedford Park, South Australia.
Coleman
M
M
Brennan
J
J
D000179:Acrylates; D001485:Basement Membrane / Q000032:analysis; D003890:Desiccation; D005615:Freezing; D006801:Humans; D007150:Immunohistochemistry; D007678:Kidney Glomerulus / Q000648:ultrastructure*; D008689:Methacrylates; D011309:Preservation, Biological / Q000379:methods*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D014885:Waxes
D000179:Acrylates; D008689:Methacrylates; D014885:Waxes; C005044:hydroxyethyl methacrylate