We show in this communication that polyacrylamide gel dcctrophoresis (PAGE) and immunoblotting ofproteins can be performed using one to two 5-7-tm paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alchohol, or modified Carnoy's solution). Proteins for study were cxtracted from paraffin sections of mouse foot pad and liver. The presence ofunaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immu

Experiments from different laboratories have shown that benzoyl peroxide (BzPo) and other organic peroxides are effective tumor promoters in the mouse skin two-stage carcinogenesis system. In the present paper we have studied the short-term effect of six other organic peroxides, which have not been previously assayed as skin tumor promoters. These compounds were chosen for their molecular diversity, the type of radical predicted to be formed, solubility and availability. The parameters evaluated in this study include a series of short-term markers of tumor promotion, hyperplasia, induction of dark basal keratinocytes and induction of ornithine decarboxylase activity. After single applications the biological activity of the compounds was: m-chloroperoxybenzoic acid greater than di-m-methylbenzoyl peroxide greater than dicumyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)mono-peroxycarbonate greater than benzoyl peroxide greater than di-m-chlorobenzoyl peroxide greater than di-t-butyl peroxide greater than t-butyl hydroperoxide. After multiple applications, the order of activity of the compounds was: dicumyl peroxide greater than di-m-methyl-benzoyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)monoperoxy carbonate greater than m-chloroperoxybenzoic acid greater than di-m-chlorobenzoyl peroxide greater than t-butyl hydroperoxide greater than benzoyl peroxide greater than di-t-butyl peroxide. The difference of activity among the different compounds did not seem to correlate directly with the chemical stability of the compound; it is more likely that the activity depends on different factors such as percutaneous absorption, metabolism, and the rate of free radical formation in vivo. The data presented here further support the association between free radicals and tumor promotion since all of the compounds, with the exception of one, were active in inducing the short-term markers of tumor promotion. It will also establish conditions for future tumor experiments.

SUMMARYImmunochemical and immunocytochemical techniques have been used to characterize viral glycoproteins and endogenous rat leukaemia viruses (RaLV) produced both by Novikoff hepatocellular carcinoma cells and spontaneously transformed Wistar rat embryo cells (WRC). Results from immunocytochemical analysis demonstrated that RaLV produced by Novikoff and WRC cells could be distinguished by their unique patterns of reactivity with xenoantisera raised against virus particles or viral glycoproteins. This differential labelling was unexpected since all the antisera tested had been shown by immunoprecipitation and immunodepletion analysis to be reactive with viral glycoproteins expressed on the cell surface. Since no significant differences in cell surface-associated viral glycoproteins and those shed from the cell surface were detected by pulse iodination analysis, it was concluded that the apparent discrepancy between immunoferritin labelling and immunoprecipitation analysis resulted from differences in antigen accessibility on intact virions caused by structural differences in the viral glycoproteins expressed on Novikoff and WRC cells. This conclusion was supported by results from ferritin-lectin labelling, affinity chromatography and neuraminidase digestion studies which demonstrated differences in the saccharide moieties on both virion and cell surface-associated viral glycoproteins. Further evidence of structural differences was provided by limited digestion with trypsin and V8 protease of the M~ 64 000 (Nov gp64) and Mr 68 000 (W RC gp68) viral glycoproteins immunoprecipitated from Novikoff and WRC cells, respectively, with either monospecific anti-Rauscher murine leukaemia virus anti-gpT0 serum or monospecific antiserum against Nov gp64 (anti-gp64). Results from digestion studies showed that all the major cleavage fragments from WRC gp68 were of higher molecular weight than their Nov gp64-derived counterparts. Evidence that Nov gp64 and WRC gp68 both share structural homology with other murine viral gp70s was suggested by results from immunoprecipitation analysis with anti-gp70 and anti-gp64 sera under reducing and non-reducing conditions which demonstrated the presence of an interchain disulphide bond in both glycoproteins and showed that at least some of these molecules exist on the cell surface as disulphide-linked heterodimers of Mr 78000 and 82000.

An ideal fixative for electron microscope cytochemical studies would adequately preserve fine structure in addition to enzymatic and/or antigenic activity of specimens. Formalin is widely used as a fixative for cytochemical studies since it nearly fulfills these criteria. However, immunoelectron microscopic studies often require prolonged incubation of specimens in serum and ferritin or peroxidase conjugate. Such treatment may result in drastic morphological alteration of specimens since formalin fixation yields weak and reversible chemical linkages. Acrolein fixation results in fine structural preservation similar to that observed following glutaraldehyde fixation. Dilute solutions of acrolein have been found suitable for fixation of cells in which certain viral antigens may be demonstrated by the immunofluorescence test.

Few immunoelectron microscopic studies have been carried out in which sera of mice bearing mammary tumors were tested for the presence of naturally occurring antibodies to MMTV. In one study it was reported that sera of mice with spontaneous mammary tumors did not contain antibodies to MMTV tested by the direct immunoferritin method. It was recently reported that sera of mice bearing spontaneous mammary tumors and at least some tumor- free mice possess antibodies to MMTV which can be demonstrated by the indirect immunoperoxidase procedure. The present study was undertaken to determine (i) if naturally occurring serum antibodies to MMTV could be detected using the indirect immunoferritin method, (ii) the distribution of such antibodies in various strains of inbred mice and(iii) the degree to which immunoferritin results correlate with results derived from the same sera tested by fixed immunofluorescence.