Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver

Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 m in thickness. For all the enzymes that could be detected, the 6 m : 3 m ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.     

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 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 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 histochemistry of rat mast cell tryptase

Fixation and staining conditions for rat mast cell tryptase and its histochemical distribution in different rat tissues were investigated. Prostate, skin, lung, gut, stomach and salivary glands were fixed in either aldehyde or Carnoy fixatives and then frozen or embedded in paraffin wax. Preservation of tryptase enzymic activity against peptide substrates required aldehyde fixation and frozen sectioning. Of the peptide substrates examined, z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthylamide proved the most effective for the demonstration of tryptase. Double staining by enzyme cytochemistry followed by immunological detection of tryptase showed that, in all tryptase-containing mast cells, the enzyme is at least in part active. Conventional dye-binding histochemistry was used to confirm the identity of mast cells. Aldehyde-fixed mucosal mast cells required a much shorter staining time with Toluidine Blue if tissue sections were washed directly in t-butyl alcohol. Double staining by enzyme cytochemistry and dye binding showed that tryptase is absent from mucosal and subepidermal mast cells, which are also smaller in size and appear to contain fewer granules than connective tissue mast cells. This study demonstrates that rat mast cell tryptase, unlike tryptases in other species, is a soluble enzyme. It is stored in an active form and is absent from some mast cell subpopulations in mucosa, skin and lung. © 1998 Chapman & Hall     

Enzyme histochemistry of the mesocoxal muscles of Periplaneta americana

Histochemical techniques have been employed to characterize enzymatic activity in the mesocoxal muscles of the cockroach, Periplaneta americana. Through our studies of the enzymes myosin-ATPase, NADH reductase, succinic dehydrogenase (SDH), and lactic dehydrogenase (LDH), we were able to classify fibers within these muscles according to criteria established for muscle fibers of vertebrates. Many of the mesocoxal muscles possess two different and distinct populations of fibers, whereas the remaining muscles are homogeneous with respect to their constituent fibers. The data presented here indicate biochemical heterogeneity for muscles of differing structural and functional features and possible neurotrophic influences upon oxidative enzymes and myosin-ATPase isozymes.     

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.     

Enzyme histochemistry of cultured ovarian cells. II. Histochemistry of porcine theca interna cells in tissue culture.

By means of histochemical technique the activities of delta53beta-, 17beta-, 20alpha-hydroxysteroid dehydrogenases and glucose-6-phosphate dehydrogenase, as well as alkaline and acid phosphatase were investigated in monolayer cultures of theca interna cells, isolated from preovulatory porcine ovarian follicles. It was found that theca interna cells exhibited high and constant activity of delta53beta-hydroxysteroid dehydrogenase and G6P-DH, whereas activities of both 17beta- and 20alpha-hydroxysteroid dehydrogenase were lower and showed some fluctuations during in vitro culture. Addition of LH to the medium brought about the increase of all studied dehydrogenases. FSH was less effective. Estradiol showed quite and inhibiting effect. All the hormones mentioned above caused the increase of alkaline and acid phosphatase activity in cultured porcine theca interna cells.     

Enzyme histochemistry of the sheep uterus during the oestrous cycle

Enzyme histochemical techniques were applied to frozen sheep uteri from different stages of the oestrous cycle. The localization and activities of succinate, lactate, glucose-6-phosphate, and isocitrate (NADP+) dehydrogenases and acid and alkaline phosphatases were studied in the luminal and glandular epithelia, caruncle and myometrium. Enzyme activity in the sections was scored on a scale of 0--5. In general the enzyme activity in the uterine caruncles and epithelia was higher than in the myometrium. The myometrium did not show any alkaline phosphatase activity and isocitrate dehydrogenase (NADP+) activity was negligible. The low activities of acid phosphatase and lactate dehydrogenase and the moderate levels of glucose-6-phosphate and succinate dehydrogenases in the myometrium were constant. The caruncular tissue showed high levels of phosphatases and glucose-6-phosphate dehydrogenase, moderate levels of lactate and succinate dehydrogenases, and low levels of isocitrate dehydrogenase (NADP+) throughout the oestrous cycle. Much lower phosphatase and isocitrate dehydrogenase (NADP+) levels were found in the epithelium of deep glands compared with superficial glands. The high activity of acid and alkaline phosphatases in the luminal epithelium and the superficial glands was constant from mid-cycle to ovulation, but a significant decrease was observed immediately after ovulation. The level of dehydrogenases in epithelia was generally high and did not change during the oestrous cycle.     

Enzyme histochemistry on bone marrow sections after embedding in methacrylate at low temperature

Summary This paper presents a method for the application of light microscopy to enzyme histochemistry on semi-thin sections of non-decalcified bone marrow cylinders (4×15 mm), entire rat femurs and larger soft-tissue specimens (4×30 mm²) after embedding in a methacrylate mixture which is then polymerized at 4°C. The best results were obtained using 1–4 h fixation in 4% formaldehyde solution in 0.1 M cacodylate buffer and propanol, . Dehydration of the tissue in concentrated solutions of propanol was complete within 2 h. It was then impregnated for 4 h in the embedding medium containing 55% 2-hydroxyethyl methacrylate, 27% methyl methacrylate, 9% 2-hydroxyethyl acrylate, 9% propanol and 2% di-benzoyl peroxide. For the final polymerization the amount of peroxide was reduced to 0.4%, and 0.1% N,N-dimethylaniline was added as a co-initiator. Polymerization lasted about 10 h at 4°C; the heat of the reaction caused the internal temperature to rise, reaching a peak of 20°C after 6 h. The blocks could then be inserted directly into the tissue-holder of a rotation microtome and cut with a steel knife. Semi-thin sections (1–3 m), free from wrinkles, were easily obtained: on the surface of 1% acetic acid at 35°C, even bone-containing sections spread out spontaneously. Enzyme activity was well preserved throughout the entire section when tested for acid (acPase) and alkaline phosphatase (alkPase), non-specific esterase (nEst), butyrate esterase (bEst), -naphthol-AS-D-chloroacetate esterase (chEst), and peroxidase (poAse) on entire rat femurs and bone marrow biopsy cylinders of different species including human. Enzyme activity was still present in the blocks after a 2,5-years storage time. AlkPase outlined a network of reticulum cells, and marked osteoblasts and granulocytic cells (human). AcPase activity was strong in osteoclasts, macrophages, and Golgi zones of megakaryocytes and myeloid precursors. Best clearly marked monocytes and fat cells (rat), but not bone marrow macrophages, nEst followed the pattern of AcPase. PoAse was seen in the osteolytic rim of osteoclasts and in granulocytes. Treatment of the sections with 20% sucrose prior to incubation broke the latency of acPase and alkPase after overfixation.