Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.     

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary Improved histochemical multi-step techniques for the demonstration of glucose 6-phosphate isomerase and phosphoglucomutase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the glucosephosphate isomerase converts the substrate d-fructo-furanose 6-phosphoric acid to d-gluco-pyranose 6-phosphoric acid, and the phosphoglucomutase converts the substrate -d-glucose 1-phosphate to the same reagent, which in turn is oxidized, by exogenous and endogenous glucose 6-phosphate dehydrogenase to d-glucono--lactone 6-phosphoric acid. Concomittantly the electrons are transferred via NADP⁺, phenazine methosulphate and menadione to nitro-BT. Sodiumazide and amytal are incorporated to block electron transfer to the cytochromes.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary An improved histochemical technique for the demonstration of acid phosphatase in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of enzyme into the medium during incubation. Moreover fixation of the tissue sections in order to minimize enzyme diffusion and that causing a partial inactivation of the enzyme, is no longer necessary. In the histochemical system the enzyme catalyzes the hydrolyzes of naphthol AS-BI phosphoric acid. The enzyme localization is visualized by means of simultaneous coupling of the released naphthol with hexazotized pararosanilin. Problems involved in the histochemical demonstration of the enzyme are discussed.This investigation was in part supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO).The author wishes to acknowledge the valuable technical assistance of Mr. E. D. J. Lindenbergh and Mr. A. H. T. Vloedman.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary An improved histochemical technique for the demonstration of lactate dehydrogenase activities in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of lactate dehydrogenase into the medium during incubation. In the histochemical system the NAD⁺-dependent enzyme catalyzes the electron transfer from lactate into NAD⁺. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Amytal is incorporated into the incubating-medium to block electron transfer to the cytochromes. Problems involved in the histochemical demonstration of lactate dehydrogenase activity are discussed.This investigation was in part supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Enzyme histochemical studies on ATPase activities in longitudinal sections of striated muscle tissue in the rat

Synopsis It has been found in this study that ATPase activity is unequally distributed along the individual fibres of the soleus in the rat and occurs with an irregularity that may influence evaluations of enzyme activities in individual fibres if performed solely on transverse-sectioned tissue. The tests on the longitudinal sections have demonstrated that classification of individual fibres in a transverse-section is dependent on which parts of the fibres are included in the section and which, in turn, may not necessarily reflect thegeneral activity of the fibres. Possible geometric artefacts are discussed.     

Enzyme histochemical differentiation of white adipose tissue in the rat

Subcutaneous adipose tissues from fetal and young rats were studied with enzyme histochemical techniques. Lipid staining and histological evaluation were also utilized to compare the development of a wide variety of enzyme activities to cytoplasmic lipid deposition and morphological differentiation of adipocytes. Three distinct stages of adipose-tissue differentiation were postulated. In stage III, adipocytes were morphologically differentiated (rounded, basal-lamina positive) and enzyme reactive for many enzymes. In stage II, however, adipocytes were reactive for some enzymes but were not morphologically differentiated. Stage I adipose tissue was histologically distinct from connective tissue but did not contain lipid-laden cells or enzyme-reactive cells. Stages I and II (95%) were predominant in fetuses, whereas stage III (90%) was predominant in young animals. Histochemical analysis of adipocytes in newborn rats established the metabolic competence of these cells despite their small size. These studies indicate that enzymatic differentiation of adipocytes clearly precedes morphological differentiation.     

An improved histochemical method for the demonstration of disaccharidases with natural substrates

Summary A simple histochemical method for the demonstration of disaccharidases is described. The medium consists of natural disaccharides (as substrates) and glucose oxidaseperoxidase-3,3-diaminobenzidine tetrahydrochloride (as the detecting agent) buffered to pH 6.0 with 0.05 M citric acid phosphate buffer in 1/2% agar gel. This method which enables the localization of disaccharidases on the histological level is very suitable for the routine assessment of activities of disaccharidases in enterobiopsies.     

A new direct lead technique for histochemical demonstration of alkaline phosphatase activity.

A lead method for demonstrating alkaline phosphatase is described. The method is based on direct precipitation of lead as lead phosphatase at pH 9.5, the pH optimum of the enzyme. Stable incubation medium was achieved by using tartrate, instead of maleate, as chelating for lead. The method was found to be suitable for visualization of alkaline phosphatase in different types of tissues.     

Enzyme histochemical studies on rat amygdala in fetal alcohol syndrome

Histochemical studies were made of the activity of oxidative and hydrolytic enzymes in rat amygdala in FAS (Foetal Alcohol Syndrome). Ethanol in a dose of 9 g/kg/day was administered to rats during pregnancy and lactation in 6% aqueous solution as the only available liquid. The control rats received an equivalent diet in which ethanol was substituted by an equicaloric amount of sucrose. The offsprings were examined at the end of the 6th postnatal week. The activity of the lysosome and membrane enzymes, as well of some enzymes participating in the neurotransmission, was changed. A different decrease of the activity of oxidoreductases of Krebs cycle, glycolysis and pentose cycle was observed. The changes in the enzyme activity in the amygdala in FAS suggest alterations in the metabolism of the nervous tissue, rather than structural damages of cell organelles.     

An improved method for determining the activity of benzoyl-arginine p-nitroanilide-hydrolyzing enzyme in crude tissue extracts

The method described in this communication is sensitive and allows the direct determination in crude tissue extracts of the enzyme that hydrolyzes α-N-benzoyl-dl-arginine p-nitroanilde (Bz-Arg-NPhNO₂). It may be useful in determining cathepsin B activity in crude enzyme preparations when the reaction mixture is incubated with an inhibitor of trypsinlike enzymes which are also capable of hydrolyzing Bz-Arg-NPhNO₂.     

Polyacrylamide gel technique for the histochemical demonstration of soluble enzymes.

Soluble enzymes were immobilized and visualized by polyacrylamide gel slabs, impregnated with the incubation medium including auxiliary enzymes. The method has several advantages over existing techniques which make use of gel films or a semipermeable membrane. The diffusion of tissue compounds is effectively limited, while auxiliary enzymes may be operative. Moreover the viscosity of the medium is temperature-independent so that the incubation temperature can be varied. To demonstrate the suitability of the method glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, phosphoglucomutase and aldolase were visulaized in human or rat skeletal muscle. Cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase were both visualized in the absence of added NAD+ and menadione. For the visualization of ATP producint enzymes, like creatine kinase and pyruvate kinase, the method is not suitable.