Detection of peroxisomal proteins and their mRNAs in serial sections of fetal and newborn mouse organs

We present a protocol for detection of peroxisomal proteins and their corre sponding mRNAs on consecutive serial sections of fetal and newborn mouse ti ssues by immunohistochemistry (IHC) and nonradioactive in situ hybridizatio n (ISH). The use of perfusion-fixation with depolymerized paraformaldehyde combined with paraffin embedding and digoxigenin-labeled cRNA probes provid ed a highly sensitive ISH protocol, which also permitted immunodetection wi th high optical resolution by light and/or fluorescence microscopy. Signal enhancement was achieved by the addition of polyvinyl alcohol (PVA) for ISH color development. For IHC, signal amplification was obtained by antigen r etrieval combined with biotin-avidin-HRP and Nova Red as substrate or by th e catalyzed reporter deposition of fluorescent tyramide. Using this protoco l, we studied the developmental changes in localization of the peroxisomal marker enzymes catalase (CAT) and acyl-CoA oxidase 1 (AOX), the key regulat ory enzyme of peroxisomal beta -oxidation, at the protein and mRNA levels i n mice from embryonic Day 14.5 to birth (P0.5). The mRNA signals for CAT an d AOX were detected in sections of complete fetuses, revealing organ- and c ell-specific variations. Here we focus on the localization patterns in live r, intestine, and skin, which showed increasing mRNA amounts during develop ment, with the strongest signals in newborns (P0.5). Immunolocalization of the corresponding proteins revealed, in close correlation with the mRNAs, a distinct punctate staining pattern corresponding to the distribution of pe roxisomes.