To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we studied the effects of control, fish oil, and safflower oil diets on peroxisomal content, fatty acyl-C...

/pdf/contrasting-effects-of-fish-oil-and-safflower-oil-on-hepatic-3incw7a1m0.pdf

Contrasting effects of fish oil and safflower oil on hepatic peroxisomal and tissue lipid content

There have been changes in the practice of fixation over the past 10 years. There seem to be at least two different pressures working. On the one hand, there is increasing diversity in cell biological techniques which in turn demands more variable fixation procedures. Some of these have been outlined. Some of this change in practice has percolated through to pathology where it has been found to be diagnostically useful. In surgical pathology on the other hand, there is the continuing financial pressure for more rapid through-put of specimens which includes more rapid fixation, often with the loss of potential for subsequent chemical investigations. These are the horns of the dilemma; both are wanted at the same time. It seems that there is no magical fixative in sight which will permit all investigations on all tissues. Rather, it seems that the future will hold increasing diversity in fixation procedures which will demand that practitioners be well informed as to possibilities which, hopefully, may lend to better understanding of the problems and mechanisms of fixation.

Cell and tissue fixation, 1972-1982.

In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.

Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine.

Effects of 17a-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish

The morphological alterations of hepatocytes of female zebrafish, Brachydanio rerio, and fingerling rainbow trout, Salmo gairdneri, following prolonged exposure to 0.04, 0.2 and 1 mg/L of 4-chloroaniline were investigated by means of light and electron microscopy. Changes in peroxisomes were visualized by cytochemical demonstration of catalase activity after incubation in the alkaline diaminobenzidine medium. The amount of storage products was illustrated by the silver impregnation technique. In a dose-dependent manner, the reaction of female zebrafish liver is characterized by a disturbance of hepatocytic compartmentation, progressive fenestration and fractionation of the rough endoplasmic reticulum (RER), a decrease in the number of peroxisomes and catalase activity, stratified inclusions in mitochondria, and an augmentation of lysosomes and myelinated bodies. Trout hepatocytes display nuclear inclusions, fractionation and vesiculation of the RER, and an increase in mitochondria, but a decrease of peroxisomes and catalase activity. Whereas glycogen stores are exhausted at 1 mg/L 4-chloroaniline, lipid deposits are amplified. An elevated rate of hepatocytic mitosis as well as the occurrence of glycogen-condensing cells probably derived from hepatocytes indicate the induction of proliferative processes in trout liver. Evaluation and comparison of results with earlier reports suggest that despite the unspecificity of some alterations the combination of pathological symptoms yields a syndrome specific of the species and the substance studied. As a consequence, histological and cytological investigations are recommended as a routine supplement in an integrated test schedule for the assessment of sublethal effects of pollutants in the aquatic environment.

Species-specific reaction of liver ultrastructure in zebrafish (Brachydanio rerio) and trout (Salmo gairdneri) after prolonged exposure to 4-chloroaniline

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° 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° C) the maximal pigment production was obtained at pH 10.5 but incubation at 45° 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.

https://zellbiologie.uni-bonn.de/arbeitsgruppen-1/prof. herzog/24.-lehir-et-al.-cytochemical-detection-of-catalase.histochemistry-64-1979.pdf

M. LeHir

Papers

Cytochemical detection of catalase with 3,3'-diaminobenzidine. A quantitative reinvestigation of the optimal conditions.