Current cytochemical techniques for the investigation of peroxisomes: A review

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorde rs, with further deepening of our understanding of the metabolic role of th is ubiquitous cell organelle. There have been many recent reviews on bioche mical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the st ate-of-the-art cytochemical techniques available for investigation of perox isomes. After a brief introduction into the use of the 3,3'-diaminobenzidin e method for localization of catalase, which is still most commonly used fo r identification of peroxisomes, the cerium technique for detection of pero xisomal oxidases is discussed. The influence of the buffer used in the incu bation medium on the ultrastructural pattern obtained in rat liver peroxiso mes in conjunction with the localization of urate oxidase in their crystall ine cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeli ng by automatic image analysis enables quantitative assessment of alteratio ns of proteins in the matrix of peroxisomes. This provides a highly sensiti ve 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 p reembedding immunocytochemistry for identification of ER-derived early pero xisomes is emphasized. The use of GFP expressed with a peroxisomal targetin g signal for the investigation of peroxisomes in living cells is briefly di scussed. Finally, the application of for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol oxidase using perfusion-fixati on, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mR NAs encoding peroxisomal proteins.