Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections

Summary The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study whow that the enzyme histochemical technique — except for some limitations — is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study show that the enzyme histochemical technique--except for some limitations--is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

Thiosemicarbazide-ferricyanide reduction for the histochemical demonstration of aldehydes in tissue sections

Aldehydes produced from carbohydrates by oxidation or acid hydrolysis may be visualized by application of aqueous thiosemicarbazide followed by Schmorl's ferricyanide reduction. The thiosemicarbazide reacts with the aldehydes by its hydrazine group, while its thiocarbamyl group remains active. The thiocarbamyl moiety is a strong reducing group that converts ferricyanide to ferrocyanide in Schmorl's reaction. The ferrocyanide is trapped immediately by the ferric salt, which deposits Prussian blue at the site of the aldehydes thereby demonstrating the location of the original substance.     

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

Summary The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25°C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25°C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.     

Cytospectrophotometric study of the kinetics of histochemical reaction of GABA-transaminase in cryostat sections of rat cerebellar cortex

Summary A modification of the histochemical method for demonstration of GABA-transaminase is proposed. Substrate and cofactor concentrations are chosen on the basis of a kinetic investigation in cryostat sections of the rat cerebellar cortex. Enzymatic reactions were measured by quantitative microspectrophotometry. Michaelis constants for -oxoglutarate in the Purkinje cell layer and granular layer (K_m 1.7×10^–3 M) and white matter (K_m 3,8×10^–3 M) are found. It is shown that -oxoglutarate in concentrations higher than 5.2×10^–3 M (1 mg/ml) suppresses the reaction in sections by competitive inhibition. The advisability of addition of malonate, PMS and cyanide to the incubation medium is confirmed. It is suggested that there are some isoenzymes of GABA-transaminase with predominant localization either in neurons or glia.     

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 reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10 degrees). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out successfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azo-coupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative histochemical study of the reaction kinetics to inosine-5-monophosphate dehydrogenase and 5'-nucleotidase in cryostat sections of rat liver

The data are presented on kinetics of histochemical enzymatic reactions demonstrating two enzymes taking part in the purine metabolism--inosine-5-monophosphate dehydrogenase (IMPD) and 5'-nucleotidase (5'-NT). It is shown that IMPD has a very weak affinity to substrate IMP (KM = 2.5 . 10(-2) M); this fact partially explains the low rate course in cryostat sections. Cytosol and membrane forms of 5'-NT have the maximum affinity to AMP (KM for membrane and cytosol forms being 1 . 10(-3) M and 2.1 . 10(-3) M, resp.). When IMP is used as a substrate, 5'-NT localized in cytosol has much lower KM as compared to the membrane form. Cytoplasmic 5'-NT is thermostable. It is suggested that a low rate of histochemical reaction demonstrating IMPD is caused by a degradation of substrate by the membrane form of 5'-NT, the Michaelis constant of which is 5 times less than that of IMPD.     

Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

Summary The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10°). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out succesfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azocoupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix. Changes of enzyme activities in synoviocytes, chondrocytes and osteoclasts during induced arthritis are discussed.     

Optimal parameters for the histochemical demonstration of acetylcholinesterase in plastic sections of rat brain

A modified method for improved preservation and optical resolution of acetylcholinesterase (AChE)-containing structures in adult rat brain is described. Optimal tissue preparation included fixation in paraformaldehyde 4%, glutaraldehyde 0.1%, and sucrose 7% in 0.1M Sorensen's phosphate buffer, pH 7.4, rinsing in buffer 50 mM with respect to NH4Cl and 2% with respect to sucrose, acetone dehydration, vacuum infiltration with LKB Historesin, and polymerization at 4 C, overnight incubation of 10 microns sections at 37 C in the AChE histochemical reaction mixture and silver intensification according to Hedreen et al. Demonstration of AChE enzyme activity in the cholinergic projection from the rat basal forebrain to the ipsilateral hippocampus exemplifies the usefulness of the technique. The method provides an excellent demonstration of AChE-positive axonal processes and enables the pharmacohistochemical visualization of cholinergic neurons. This procedure offers a convenient method for analysis of cholinergic neurons that avoids potential artifacts inherent in other AChE histochemical procedures.