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.     

Alternative myelomonocytic differentiation of HL-60 reflects dual prospective potency of promyelocytes in human

The permanent promyelocytic cell line HL-60 was subjected to stimulation with dimethyl sulfoxide (DMSO) and retinoic acid (RA), as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and lymphokine conditioned media for the induction of granulocytic or monocytic differentiation, respectively. Cells were investigated cytochemically using alpha-naphthylacetate esterase (acid esterase; AcE), naphthol AS-D chloroacetate esterase, and peroxidase reactions. In addition, the granulocyte or monocyte specific isoenzyme patterns of AcE as an intracytoplasmic property and the immunoreactivity to monoclonal antibodies recognizing granulocytes and monocytes (Ki-M2, Ki-M5) or monocytes alone (Ki-M1) were considered. The results indicated that HL-60 cell line bear the potency to evolve into granulocytes as well as monocytes. Additional studies performed on normal human bone marrow stained for AcE led to the conclusion that the myeloid cell line remains bipolar until the maturation stage of promyelocytes. Myelocytes being AcE positive only in 11.5 +/- 5.0 are heterogeneous and display the first indications of separated monocytic or granulocytic differentiation.     

Fluoride resistant alpha-naphthyl acetate esterase and combined enzyme histochemistry in the study of normal and pathologic lymphoid tissues

Alpha-Naphthyl acetate esterase (ANAE) and fluoride resistant alpha-naphthyl acetate esterase (FRANAE) have been compared as histochemical methods to identify T lymphocytes in sections of normal and pathological human lymphoid tissues. In addition, the FRANAE method was combined with alkaline phosphatase (ALP) in order to simultaneously evaluate the relationship between T lymphocytes and fibroblastic reticular cells (ALP) positive). The "dot like" esterase positivity of T lymphocyte was better evaluated by using FRANAE when compared to ANAE because of fluoride inhibitor of the strong esterase activity of dendritic cells and most macrophages. The combined ALP-FRANAE method clearly demonstrated a large number of fibroblastic reticular cells within the T-areas in various normal and pathological tissues such as hyperplastic lymph nodes and especially in the lymph nodes and spleens from patients with Hodgkin's disease.     

Histochemical evidence of three types of esterases in the adrenal medulla of the rat

Summary Esterases of the adrenal medulla have been studied histochemically using alpha-naphthyl acetate and butyrate as substrates, Blue RR Salt as a coupler and eserine and E, 600 as inhibitors. Three types of esterase activity were thus demonstrated: (1) cholinesterase activity in the nerve fibres, ganglion cells and secretory medullary cells; (2) eserine resistant but E. 600 sensitive esterase activity in the ganglion cells and secretory cells; (3) E. 600 resistant activity in strongly positive, unidentified cells scattered in the medulla. The histochemical picture was essentially similar in sections of formalin-fixed tissue and in fresh sections subjected to the voltage gradient employed for electrophoretic separation of esterases. It is concluded that esterases histochemically demonstrable in sections are desmo-enzymes and at least to a major part different from the lyo-enzymes which can be separated by starch gel electrophoresis.With 6 Figures in the Text     

Immunobiology of the human placenta: II. Localization of macrophages,in vivo bound IgG and C3

Human placentae ranging in gestational age from less than 4 weeks to full term were studied for the localization of IgGFc receptor bearing cells by the EA adsorption method. Most cells populating the area beneath the syncytiotrophoblast in trophoblastic villi, e.g., the stromal region, adsorbed EA. When cell suspensions of trophoblastic tissue were prepared and the individual cells were rosetted and characterized, most IgGFc receptor bearing cells were determined to be macrophages by morphology, phagocytosis and nonspecific esterase staining. A few of the syncytiotrophoblast cells also were IgGFc receptor positive. When esterase staining was applied to sections of trophoblastic tissue, the pattern of esterase positivity paralleled the pattern of EA adsorption in that most cells in trophoblastic villi were positive while decidual cells were negative. Furthermore, the pattern of IgG bound in vivo as determined by direct immunofluorescence paralleled the EA adsorption and esterase staining patterns. The bound IgG was removed by prolonged washing of the trophoblastic tissue at 37 °C which suggested that most IgG was bound to IgGFc receptors in trophoblastic villi. The results are discussed with respect to the possible functions of large numbers of macrophages located at the interphase between maternal and fetal circulations.     

Improvement in the Histochemical Demonstration of Acid Phosphatase, β-Galactosidase and Nonspecific Esterase in Glycol Methacrylate Tissue Sections by Cold Temperature Embedding

A simple, cold-embedding method (on cracked ice at 2 C) is presented for the demonstration of acid phosphatase, β-galactosidase and nonspecific esterase in glycol methacrylate tissue sections.     

STUDIES ON ENZYMATIC HISTOCHEMISTRY : XXVI. THE HISTOLOGICAL DISTRIBUTION OF CHOLINE ESTERASE IN THE GASTRIC MUCOSA NORMALLY AND AFTER ADMINISTRATION OF CERTAIN DRUGS

Extraction experiments demonstrated that choline esterase could be removed from microtome sections of tissue with as great facility by 0.9 per cent NaCl as by 30 per cent glycerol. The quantitative distribution of choline esterase through the wall of the pig stomach was studied, and it was found that the epithelial cell region possessed the greatest activity and muscle tissue the least. Pylorus was more active than fundus or cardia. The enzyme activities found were independent of the physiological state of the normal stomach at the time the animal was killed. Neither intramuscular injection of acetyl choline, eserine, nor atropine shortly before killing had significant influence upon the activity in any region of the stomach. The implications of these results were discussed.     

QUANTITATIVE ESTIMATION OF LYO- AND DESMOENZYMES IN TISSUE SECTIONS WITH AND WITHOUT FIXATION

1. Tissue sections eight microns thick were exposed to various experimental conditions used in histochemistry, and the effect upon the activities of esterase, the phosphatases, leucine aminopeptidase, β-glucuronidase, and arylsulfatase was determined colorimetrically. 2. Significant differences were found in the amounts of the lyo and desmo fractions of these enzymes. The desmo components were found to be for esterase, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, β-glucuronidase, and arylsulfatase, ⅓, 2/3, 2/3, ½, ⅛, and ⅛ of the total enzymatic activity respectively. 3. Variations in the time and in the temperature at which diffusion was studied and of the pH and salt concentration of the solution into which the sections were placed, resulted in differences in the amount of enzymatic activity which remained in the tissue section. Some enzyme loss by diffusion was noted even after fixation of the tissue section. 4. The significance of the findings with respect to some of the concepts of localization of enzymes in tissue sections was discussed.     

Complete Staining of Neuromuscular Innervation with Bromoindigo and Silver

Frozen sections of musde fixed in buffered formaldehyde (pH 7.3) are first incubated for localization of esterase using 5-bromoindoxyl acetate as substrate. The sections are then mounted on slides and stained by a urea-silver nitrate method for axons. Result: subneural apparatus—blue; axons—black; other tissue components in various shades of grey.     

Histochemical identification of human T lymphocytes from paraffin sections

The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.     

Enzymatic histochemistry of mouse kidney in plastic

Two-micrometer sections of methacrylate-embedded kidney were used to investigate the enzymatic activities of mouse kidney where the proximal tubule and Bowman's capsule from the same corpuscle were viewed in the same section. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, alpha-naphthyl butyrate esterase, and adenosine triphosphatase activities were observed in the proximal tubule, but only 5'-nucleotidase, alpha-naphthyl butyrate esterase, and alkaline phosphatase were observed in the squamous portion of the parietal epithelium of Bowman's capsule. The use of methacrylate-embedded tissue allowed more precise localization of enzymatic activity than is possible with most frozen sections. This may provide interesting applications not only for characterization of kidney diseases but also for characterization of other normal and abnormal tissues.     

Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.     

An improved method for the simultaneous demonstration of mRNA and esterase activity at the human neuromuscular junction

The aim of this study was to develop a simple means of studying the distribution of mRNA coding for post-synaptic proteins at the human neuromuscular junction. A reliable method by which to identify the junctions in tissue sections after in situ hybridization was essential. A method is described for combining the histochemical demonstration of esterase activity at the neuromuscular junction with autoradiographic localization of mRNA by in situ hybridization in the same cryostat section of skeletal muscle. The indigogenic esterase method of Strum and Hall-Craggs (1982) was modified in such a way that it is able to survive the multiple steps involved in in situ hybridization and autoradiography. The protocol is simple and reproducible and has been used successfully on sections of both rat and human skeletal muscle. To demonstrate the method, sections were reacted to reveal esterase activity and were then processed for in situ hybridization using a 35S-labelled probe specific for the -s ubunit of the acetylcholine receptor. The reaction product was retained after the lengthy in situ hybridization and autoradiographic procedures. To our knowledge, this is the first demonstration of acetylcholine receptor mRNA by in situ hybridization at human neuromuscular junctions. © Chapman & Hall     

Elastases from human and canine granulocytes, II. Interaction with protease inhibitors of animal, plant, and microbial origin.

Inhibitors of animal, plant, and microbial origin were tested against human and canine granulocytic elastases. The trypsin-chymotrypsin inhibitors from dog submandibular glands, from soybeans (Bowman-Birk) and from chickpeas show strong interaction with these proteases (Ki = 10(-8) - 10(-9)M). The trypsin-kallikrein inactivator of bovine organs (Trasylol) is not active against granulocytic elastases or against human granulocytic cathepsin G. Elastatinal, a specific inhibitor of elastases, isolated from actinomycetes (Streptomyces griseoruber), forms stable complexes with elastase from human (Ki = 6.2 X 10(-6)M) and canine granulocytes (Ki = 1.1 X 10(-6)M). A possible therapeutic application of these inhibitors for the inactivation of granulocytic proteases, which are able to degrade connective tissue in different pathological states, is discussed.     

Naphthol AS D chloroacetate esterase-positive macrophages in the cortico-medullary zone of the normal rat thymus

Naphthol AS D chloroacetate esterase (NASDCE)-positive macrophages are positioned in the cortico-medullary zone (CMZ) of the normal rat thymus. These cells contain the very strongly NASDCE-positive granules of varying size in the cytoplasm. An identical distribution within the thymic parenchyma and an identical morphological appearance is observed in CMZ macrophages after staining with aldehyde fuchsin. The incubation of thymic sections in 10% formalin at pH 7.0 for 48 h does not inhibit the activity of NASDCE in CMZ macrophages. The activity of nonspecific esterase is almost totally abolished by this treatment: only the single, largest globular inclusion within some of the cells remains weakly or moderately positive. The granular content of the CMZ macrophages does not stain metachromatically with toluidine blue and these cells are endogenous peroxidase-negative. NASDCE-positive thymic macrophages are easily distinguished from: a) NASDCE-positive mast-cells, which are confined to the capsular and septal connective tissue and contain densely packed metachromatic granules, and b) NASDCE-positive neutrophilic granulocytes, which have a specific morphological appearance and show very strong endogeneous peroxidatic activity.     

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.     

Histochemical Demonstration of Enzyme Activities in Plastic and Paraffin Embedded Tissue Sections

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, β-galactosidase, and cytochrome oxidase in plastic embedded and routine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 μm, were far superior to frozen Sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.     

Glycol methacrylate embedding in enzyme histochemistry: Application to arthropode tissue incubated for demonstration of unspecific esterase and succinic dehydrogenase activity

Summary The final products of unspecific esterase and succinic dehydrogenase were demonstrated in 1–2 m sections of tick salivary glands embedded in glycol methacrylate (GMA). In the esterase experiments, the tissue specimens were incubated after fixation in glutaraldehyde or acroleïn, and then embedded in GMA. For demonstration of succinic dehydrogenase activity, the specimens were incubated prior to glutaraldehyde fixation followed by embedding in GMA. In sections of all preparations intense enzymatic reaction was observed. High resolution light microscopy could efficiently be used for precise locating of the enzymic products, due to excellent morphologic reference in semithin GMA sections.Supported by the Deutsche Forschungsgemeinschaft. Presented in part at the 4th International Congress on Protozoology, Clermont-Ferrand (France), 2.–9. September 1973.The author was fellow of the Deutsche Forschungsgemeinschaft at the Department of Anatomy, Medical School Hannover, Federal Republic of Germany, during part of this study.     

Cytochemical studies in the blastic transformation of chronic granulocytic leukaemia

The cytochemical features of blast cells were studied in 45 patients with blastic phase of chronic granulocytic leukaemia. Various degrees of Sudan black B positivity was characteristic of myeloblastic transformation (23 patients), while in the medullary blast cells of nine patients with myelomonocytic transformation the alpha-naphthyl-acetate esterase showed intensive activity. In two cases the demonstrability of beta-thromboglobulin and factor VIII-related antigen in blast cells showing otherwise PAS, acid phosphatase and alpha-naphthyl-acetate esterase activity referred to megakaryocytic transformation. In six patients with lymphoid blast crisis proliferation of the Sudan negative blast cells with different granular PAS, acid phosphatase and/or beta-glucuronidase positivity was demonstrated. In five cases the cytochemical findings of leukaemic cells indicated biphenotypic and mixed transformation, respectively.