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Journal of Histochemistry and Cytochemistry, Vol. 49, 155-164, February 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Detection of Peroxisomal Proteins and Their mRNAs in Serial Sections of Fetal and Newborn Mouse Organs

Markus Grabenbauera, H. Dariush Fahimia, and Eveline Baumgarta
a Department of Anatomy and Cell Biology, Division of Medical Cell Biology, University of Heidelberg, Heidelberg, Germany

Correspondence to: H. Dariush Fahimi, Dept. of Anatomy and Cell Biology, U. of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany. E-mail: H.Dariush.Fahimi@urz.uni-heidelberg.de

We present a protocol for detection of peroxisomal proteins and their corresponding mRNAs on consecutive serial sections of fetal and newborn mouse tissues by immunohistochemistry (IHC) and nonradioactive in situ hybridization (ISH). The use of perfusion–fixation with depolymerized paraformaldehyde combined with paraffin embedding and digoxigenin-labeled cRNA probes provided a highly sensitive ISH protocol, which also permitted immunodetection with 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 retrieval combined with biotin–avidin–HRP and Nova Red as substrate or by the catalyzed reporter deposition of fluorescent tyramide. Using this protocol, we studied the developmental changes in localization of the peroxisomal marker enzymes catalase (CAT) and acyl-CoA oxidase 1 (AOX), the key regulatory enzyme of peroxisomal ß-oxidation, at the protein and mRNA levels in mice from embryonic Day 14.5 to birth (P0.5). The mRNA signals for CAT and AOX were detected in sections of complete fetuses, revealing organ- and cell-specific variations. Here we focus on the localization patterns in liver, intestine, and skin, which showed increasing mRNA amounts during development, 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 peroxisomes. (J Histochem Cytochem 49:155–164, 2001)

Key Words: peroxisomes, mouse development, catalase, acyl-CoA oxidase, in situ hybridization, immunohistochemistry, fluorescence microscopy


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