THE IMPORTANCE OF TISSUE FIXATION FOR LIGHT-MICROSCOPIC IMMUNOHISTOCHEMICAL LOCALIZATION OF PEROXISOMAL PROTEINS - THE SUPERIORITY OF CARNOYS FIXATIVE OVER BAKERS FORMALIN AND BOUINS SOLUTION

We have compared the effects of fixation with three commonly used fixa tives upon preservation of the antigenicity of six peroxisomal protein s in rat liver using both immunohistochemical staining acid Western bl otting of fixed tissue extracts. The immunoreactivity of all six perox isomal proteins was well preserved and peroxisomes were clearly identi fied in material fixed in Carnoy's fixative. Moreover, the correspondi ng proteins stained well in Western blots prepared from extracts of Ca rnoy-fixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins af ter fixation in Baker's formalin, but peroxisomes were still well visu alized with antibodies to catalase and some beta-oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the an tibody binding sites in Carnoy's fixed material, the avidin-biotin-per oxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminoben zidine and protein A-gold with silver intensification. Using Carnoy-fi xative and the ABC-method, we demonstrate light microscopic immunohist ochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.