Search

Peroxisomes are ubiquitous dynamic and multifunctional organelles that contribute to numerous anabolic and catabolic pathways, being essential for human health and development. Their best known functions include the oxidation of fatty acids and metabolism of hydrogen peroxide with catalase as a marker enzyme. Indeed, historically, it was the cytochemical staining of catalase in many different cells and tissues that revealed the ubiquitous presence of peroxisomes in almost all animal and plant cells. In this chapter, the method for cytochemical staining of catalase with the alkaline 3, 3'-diaminobenzidine (DAB) is described. Since aldehyde fixation is a prerequisite for staining of catalase with DAB, a method for perfusion fixation of rat liver with glutaraldehyde is presented prior to the cytochemical staining method and the subsequent tissue processing for light and electron microscopy.

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)

The intracellular localization of beta-NADPase in rat hepatocytes and Kupffer cells has been studied and compared with the pattern of TPPase in these cells. The reaction product for beta-NADPase is present in some but not all hepatocytes in two cisternae on the trans aspect of the Golgi apparatus. It is absent from the trans-most lamella and the GERL of hepatocytes. TPPase, on the other hand, is limited to the first Golgi cisterna on the trans aspect with sprinkles of reaction product in the second lamella. Considering that TPPase is a marker of the trans Golgi lamella and hepatocyte Golgi stacks contain usually 2-4 lamellae, our observations suggest that beta-NADPase is localized in the trans as well as in the intermediate Golgi lamellae of liver parenchymal cells. In Kupffer cells, the reaction product for both beta-NADPase and TPPase was found in some but not in all cells. The enzyme beta-NADPase was localized in the rigid lamella and the tubulovacuolar system of GERL. This pattern differed significantly from that for TPPase, which was found in 2-3 cisternae at the trans aspect of the Golgi complex in Kupffer cells. These observations demonstrate the difference in the localization of beta-NADPase in hepatocytes and Kupffer cells. Such differences should be taken into consideration in studies of Golgi fractions, when phosphatase reactions are used as specific markers of Golgi components.

The application of an automatic image analyzer (TAS, Leitz, Wetzlar) for determination of labeling density in protein A-gold labeled sections is described. Electron micrographs of rat liver labeled with 12 nm gold particles for peroxisomal enzymes are placed on the macrounit of TAS and the images of peroxisomes on TAS-monitor are contoured with a light pen. The instrument measures the surface of the contoured areas. Based on their gray level, the gold particles over the peroxisome are detected automatically and counted and the labeling density for each peroxisome is calculated.

Qualitative cytological criteria are concerned with the 'precision' of a histochemical reaction and form part of the general criteria for the validation of enzyme histochemical techniques. Three major problem areas are considered: (a) the general artifacts which interfere with the 'clean' appearance of the tissue sections; (b) the exact intracellular localization of the final reaction product and its relationship to specific subcellular compartments; (c) the problem of diffusion of enzymes or reaction products, or both, from their primary subcellular sites. Each of these points is discussed and illustrated by a few examples and the conclusion is drawn that by careful consideration of various criteria the enzyme cytochemical techniques can provide 'quantitative' information comparable to biochemical measurements of enzyme activity in tissue homogenates. In the example of localization of catalase with 3,3'-diaminobenzidine it is demonstrated that biochemical enzyme assays corrrelate closely with quantitative morphometric data obtained from cytochemical preparations through an automatic image analyser system.

The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5 degrees C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the "mildest" fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25 degrees C) the maximal pigment production was obtained at pH 10.5, but incubation at 45 degrees C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H2O2 in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.