Enzyme histochemistry on freeze-dried, resin-embedded tissue

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.     

Enzyme histochemistry and immunohistochemistry with freeze-dried or freeze-substituted resin-embedded tissue

Summary Freeze-drying or freeze-substitution, combined with low-temperature resin-embedding, represents a new approach to the optimum preservation of tissue for enzyme histochemistry and immunohistochemistry. This method, which avoids tissue fixation, combines excellent tissue morphology with the preservation of enzyme activity and immunoreactivity and allows high-resolution enzyme histochemical and immunohistochemical studies to be performed. The activity of a wide range of enzymes can be demonstrated in sections of freeze-dried or freeze-substituted resin-embedded tissue. Enzymes are retainedin situ with high activity, accurate localization and no diffusion. Immunohistochemical studies can also be performed on resin sections, and antigens—especially labile antigens — are immobilizedin situ without denaturation and can be demonstrated with high sensitivity and accurately localized. This method allows the localization and distribution of enzymes and antigens to be studied in relation to excellent histological and cytological detail.     

Enzyme histochemical studies on rat lungs I. An improved technique for the demonstration of enzyme activity in alveolar tissue

Synopsis Rat lungs were supported by ten different materials in an attempt to compare the quality of the sections obtained and to assess the effects of these materials on the activities of three enzyme systems in alveolar tissue as determined by histochemical techniques.Most of the supportive materials were not removed during enzyme incubation and caused some apparent inhibition of enzyme activity. Of the compounds investigated, 0.5% polyethylene glycol was found to produce the most satisfactory results with the least distortion or compression of the alveolar tissue and no apparent inhibition of the three enzymes studied, lactate dehydrogenase, non-specific esterase and alkaline phosphatase. Some of the materials investigated, notably acacia and carboxymethylcellulose, appeared to decrease the thickness of the alveolar wall. This effect was only seen in lungs supported by the most viscous materials.     

Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity

Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.     

Enzyme histochemical investigation of glycol methacrylate embedded chick embryonic tissue

Synopsis The advantages of the water-soluble glycol methacrylate (GMA) embedding procedure make it highly applicable for use with fragile early embryonic material. Not only can one obtain tissue sections containing excellent histological detail, but numerous enzymes are retained for subsequent histochemical localization. For the purpose of establishing a methodology whereby concomitant histology and histochemistry could be obtainable, various fixatives and fixation times have been evaluated on GMA embedded chick embryonic mesonephros and gonad. It was found that fixing the tissues for 1 h in a solution of 95% ethanol, 5% acetic acid and 10% neutralbuffered formalin resulted in the retention of not only excellent histology but also alkaline and acid phosphatase. Thus, with this procedure, more specific investigations of early embryonic tissue can be performed.     

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.     

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.     

Dehydrogenase enzyme histochemistry on freezedried or fixed resin-embedded tissue

Summary A method has been developed for the histochemical demonstration of a variety of dehydrogenases in freeze-dried or fixed resin-embedded tissue. Seven dehydrogenases were studied. Lactate dehydrogenase, NADH dehydrogenase and NADPH tetrazolium reductase were all demonstrable in sections of paraformaldehyde-fixed resin-embedded tissue. Freeze-dried specimens were embedded, without fixation, in glycol methacrylate resin or LR Gold resin at either 4°C or –20°C. All the dehydrogenases except succinate dehydrogenase retained their activity in freeze-dried, resin-embedded tissue. Enzyme activity was maximally preserved by embedding the freeze-dried tissue specimens in glycol methacrylate resin at –20°C. The dehydrogenases were accurately localized without any diffusion when the tissue sections were incubated in aqueous media. Addition of a colloid stabilizer to the incubating medium was not required. Freeze-drying combined with low-temperature resin embedding permits accurate enzyme localization without diffusion, maintenance of enzyme activity and excellent tissue morphology.     

NADH dehydrogenase and NADH oxidation inBacillus megaterium

Only one type (membrane-bound form) of NADH dehydrogenase could be detected in the log-phase cells ofBacillus megaterium. By sonification this enzyme could be effectively solubilized, while NADH oxidase remained bound to the membrane. A molecular weight of about 40 Kd was estimated for the dehydrogenase by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) with an activity stain. Mercuric chloride and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) were inhibitors for both the NADH dehydrogenase and oxidase inB. megaterium. The inhibition studies of NADH oxidation suggested that NADH dehydrogenase provided the primary electron source for NADH oxidase in this organismin vitro. NADH dehydrogenase was highly specific for NADH, and K_m was estimated to be 28.2 M. The enzyme was subjected to end-product inhibition of a competitive type.     

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.