IMMUNOGOLD LOCALIZATION OF TGF-BETA-1 PROTEIN AND MESSENGER-RNA IN HUMAN SKIN USING A COLLOIDAL GOLD DIGOXYGENIN SYSTEM

Tissue preservation, and immunogold cytochemical and in-situ hybridiza tion labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce opti mal preservation, protein and mRNA labelling. Nine combinations of fix ative and embedding resin were therefore studied using postembedding i mmunoelectron microscopy and a novel immunogold digoxygenin in situ hy bridization (ISH) system, to quantitate the presence of transforming g rowth factor beta 1 (TGF beta 1) protein and message in human skin. Th e best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (L A-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde a nd fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol de hydration coupled with LA-GMA embedding resulted in reasonable preserv ation. Based on quantitative measures of the labelling density for TGF beta 1 protein and mRNA, immunogold labelling was adequate with 1% gl utaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethane diol dehydration and LA-GMA embedding. ISH labelling under basal condi tions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde . The ISH label in tissue fixed with 1% glut araldehyde and embedded i n LA-GMA was significantly increased by treatment with proteinase K. O verall, ethanediol dehydration was associated with a good immunoelectr on microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% p araformaldehyde resulted in a consistently detectable ISH label. LA-GM A embedding with 1% glutaraldehyde fixation gave a good result with bo th IEM and ISH labelling.