Current Cytochemical Techniques for the Investigation of Peroxisomes: A Review

Abstract

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorders, with further deepening of our understanding of the metabolic role of this ubiquitous cell organelle. There have been many recent reviews on biochemical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the state-of-the-art cytochemical techniques available for investigation of peroxisomes. After a brief introduction into the use of the 3,3'-diaminobenzidine method for localization of catalase, which is still most commonly used for identification of peroxisomes, the cerium technique for detection of peroxisomal oxidases is discussed. The influence of the buffer used in the incubation medium on the ultrastructural pattern obtained in rat liver peroxisomes in conjunction with the localization of urate oxidase in their crystalline cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeling by automatic image analysis enables quantitative assessment of alterations of proteins in the matrix of peroxisomes. This provides a highly sensitive approach for analysis of peroxisomal responses to metabolic alterations or to xenobiotics. The recent evidence suggesting the involvement of ER in the biogenesis of "preperoxisomes" is mentioned and the potential role of preembedding immunocytochemistry for identification of ER-derived early peroxisomes is emphasized. The use of GFP expressed with a peroxisomal targeting signal for the investigation of peroxisomes in living cells is briefly discussed. Finally, the application of in situ hybridization for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol using perfusion-fixation, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mRNAs encoding peroxisomal proteins. (J Histochem Cytochem 47:1219-1232, 1999)

Reactive oxygen species detoxification by catalase is a major determinant of fecundity in the mosquito Anopheles gambiae

“…2E). The DAB assay can give a positive staining to both catalase and peroxidase activities (22). The specificity of the DAB staining for catalase was confirmed by the following criteria.…”

“…Samples were then incubated in 2.5 mM DAB (Sigma, St. Louis, MO) and 2.5 mM H 2 O 2 (Sigma) in 0.1M Tris⅐HCl (pH 10.0) for 10 min. The high pH favors catalase compared with peroxidases (22). Samples were then removed, washed in PBS, and fixed for 1 h in 4% paraformaldehyde.…”

“…2 A and B). The cellular localization of the enzyme was determined by staining ovaries 24 h after feeding by using diaminobenzidine (DAB) as a substrate (22). Catalase activity was present only in the oocyte (Fig.…”

“…(i) Peroxidase activity, determined with a peroxidasespecific substrate in ovary homogenates, was not induced as ova matured (data not shown). (ii) The eggs stained readily under pH conditions favorable to catalase (pH 10.0) but less favorable to peroxidase (22). (iii) The DAB staining was strongly inhibited by 3-amino-1,3,4-triazole (AT) (Fig.…”

In situ heparin‐induced peroxisomal reticulum and biogenesis of peroxisomes in pulmonary intravascular macrophages (PIMs) of caprine lung: an ultrastructural and cytochemical study

“…The peroxisomes reacted positively for catalase after aldehyde fixation and 3,3Ј-diaminobenzidine (DAB) staining. The DAB technique for localization of catalase, a marker of peroxisomes, is still the cytochemical method of choice in the initial morphological survey for the identification of this organelle (Fahimi and Baumgart, 2000). We also analyzed the formation of peroxisomal reticulum (PR), a transitional organelle thought to be involved in the early stages of de novo formation of peroxisomes (Yamamoto and Fahimi, 1987;Lazarow and Fujiki, 1985).…”