Immunogold localization of TGF beta 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system.
Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF beta 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF beta 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.
Slater
M
M
Department of Physiology, University of Sydney, NSW, Australia.
Mason
R S
RS
D004076:Digoxigenin; D017979:Gold Colloid; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization / Q000379:methods*; D016253:Microscopy, Immunoelectron; D012333:RNA, Messenger / Q000032:analysis*; D012867:Skin / Q000737:chemistry*; D016707:Tissue Fixation / Q000379:methods; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000235:genetics
D017979:Gold Colloid; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D004076:Digoxigenin