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 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.     

Ultrastructural localization of a lysosomal enzyme in resin-embedded lymphocytes

Summary The intracellular distribution of lysosomal enzymes in lymphocytes has previously been only poorly defined, mainly by cytochemical procedures of low resolution. In the present study we have used a post-embedding immunogold technique to identify the precise ultrastructural localization of a lysosomal enzyme, -glucuronidase, in activated lymphocytes embedded in Lowicryl K4M resin. We show that this enzyme is present in the rough endoplasmic reticulum, in the Golgi complex, and in vesicular organelles which probably include lysosomes.     

Ultrastructural localization of a lysosomal enzyme in resin-embedded lymphocytes

The intracellular distribution of lysosomal enzymes in lymphocytes has previously been only poorly defined, mainly by cytochemical procedures of low resolution. In the present study we have used a post-embedding immunogold technique to identify the precise ultrastructural localization of a lysosomal enzyme, beta-glucuronidase, in activated lymphocytes embedded in Lowicryl K4M resin. We show that this enzyme is present in the rough endoplasmic reticulum, in the Golgi complex, and in vesicular organelles which probably include lysosomes.     

Enzyme histochemistry on freeze-substituted glycol methacrylate-embedded tissue

We developed a method for histochemical demonstration of a wide range of enzymes in freeze-substituted glycol methacrylate-embedded tissue. Tissue specimens were freeze-substituted in acetone and then embedded at low temperature in glycol methacrylate resin. All enzymes studied (oxidoreductases, hydrolases) 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-substitution combined with low-temperature glycol methacrylate embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, maintenance of enzyme activity, and excellent tissue morphology.     

Quantitative enzyme histochemistry in the brain

Two main groups of quantitative methods are used in the brain to relate enzymatic processes to cellular structures, i.e. the methods of microchemistry and microscopic histochemistry. Microchemistry tries to quantify enzyme activities in very small brain regions by miniaturizing biochemical methods, whereas microscopic histochemistry applies staining procedures to tissue sections, preserving the structural relationship that is present in situ and giving topological information on the distribution of enzymes which is indispensable in structural heterogeneous tissue as is the brain. The present review deals preferentially with microscopic methods and, in particular, with scanning microphotometry (image plane scanning). Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. end-point and kinetic (continuous monitoring) measurements which are described in detail. Methods for the microphotometric demonstration of certain important dehydrogenases (isocitrate dehydrogenases, succinate dehydrogenase, NAD-linked malate dehydrogenase, glutamate dehydrogenase and glycerol 3-phosphate dehydrogenase), of cytochrome c oxidase, hexokinase and acetylcholinesterase are presented. These methods were adapted for giving optimal demonstration of enzyme activities in the rat hippocampus. The examples are given to illustrate the aptitude and possibilities of this technique in the quantification of enzymes in the complex matrix of the brain.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8–12 h at –50 to –40°C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45°C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4°C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens.

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Ultrastructural localization of cholesterol by enzyme histochemistry

Summary The histochemical localization of cholesterol using oxidized diaminobenzidine as the final reaction product was studied at the electron microscopical level and compared with the digitonin method of cholesterol localization based on cholesterol digitonide as the final reaction product. Tissue chopper sections of fixed rat adrenal glands were incubated at 37° C in a medium consisting of 0.8 units/ml cholesterol oxidase, 1.4 units/ml cholesterol ester hydrolase, 50 units/ml horseradish peroxidase, 0.5 mg/ml diaminobenzidine, 0.1% v/v Triton X-100 (or Surfal) and an endogenous peroxidase inhibitor in 0.1m phosphate buffer, pH 7.0. An electron-dense osmiophilic reaction product was observed in many lipid droplets, intracellular vesicles and focally around mitochondria. Appropriate control experiments indicated that deposition of reaction product depended on the presence of cholesterol and the necessary enzymes. Comparison studies using digitonin confirmed the presence of cholesterol in the lipid droplets, but ultrastructural distortion limited the resolution of the more discrete deposits of cholesterol such as around mitochondria. The enzyme method permits finer resolution of these discrete deposits of cholesterol than the digitonin method because it does not cause distortion of cellular ultrastructure attributed to the formation of cholesterol digitonide. The enzyme method or a combination of enzyme and digitonin enables localization of free, esterified or total cholesterol.     

Brain enzyme histochemistry following stabilization by microwave irradiation

Summary The activities of various enzymes present in brain homogenates were assayed biochemically (a) with no pretreatment, (b) following a standard microwave treatment in saline and (c) after a standard microwave treatment in formalin. All enzyme activity was lost after the microwave — formalin in treatment. Following microwave — saline treatment, the activities of alkaline phosphatase, 5-nucleotidase, isocitrate and succinate dehydrogenases were reduced. In contrast, the activities of lactate and malate dehydrogenases were unchanged, and that of acetylcholinesterase apparently increased.Analogous outcomes were seen following attempted histochemical demonstrations of these enzymes. Thus satisfactory histochemical demonstration of all enzymes was achieved (except with alkaline phosphatase, lactate and malate dehydrogenases) following the microwave-saline pretreatment. Since acid phosphatase, catalase and peroxidase were also successfully demonstrated, it seems that microwave-saline pretreatments permit both retention of sufficient enzyme activity for histochemical demonstration to occur and retention of sufficient structural integrity for critical morphological investigations. Since the failure to stain the sites of lactate and malate dehydrogenases is not due to microwave inactivation of these enzymes, their demonstration may be possible by varying the staining procedures.     

The formulation of buffers and media for enzyme histochemistry

Summary Buffer solutions and incubating media for enzyme histochemistry are discussed in terms of pH, ionic strength and buffering capacity. A specially written program is presented. This program enables (i) buffers and media of known pH and ionic strength to be formulated; (ii) the ionic strength of a buffer solution of known molarity, and (iii) the thermodynamic acid dissociation constant of a buffer substance, to be calculated.