Journal of Histochemistry & Cytochemistry

ALLEN, JOHN M.; SLATER, JUDITH J.

doi: 10.1177/9.3.221pmid: 13860550

The properties of diphosphopyridine nucleotide diaphorase and the lactic dehydrogenase-diphosphopyridine nucleotide diaphorase system in the epididymis of the mouse have been investigated by cytochemical and quantitative methods.In normal mice diphosphopyridine nucleotide diaphorase activity was homogeneously distributed within the cytoplasm of the epithelial cells of the epididymal canal. A gradient of activity existed along the duct, with the lowest levels of activity appearing in the head segments and the highest levels in the body and tail segments.The lactic dehydrogenase-diphosphopyridine nucleotide diaphorase system differed in its cytological distribution from diphosphopyridine nucleotide diaphorase. In the cells of the body portion of the epididymal canal this system showed high levels of activity in the cell apex with low levels of activity prevailing in the general cytoplasm. In other areas the system showed low levels of activity homogeneously distributed throughout the cytoplasm.Cytochemical studies indicated that the activity of diphosphopyridine nucleotide diaphorase was depressed by orchidectomy and restored by testosterone propionate administration. The activity of this enzyme was unaffected by section of the vasa efferentia (vasectomy). The activity of the lactic dehydrogenase-diphosphopyridine nucleotide diaphorase system was depressed by vasectomy and orchidectomy. Testosterone propionate restored activity of tissues from castrated animals to levels prevailing in vasectomized animals but did not re-establish the distribution pattern observed in normal animals.Quantitative determinations confirmed and extended cytochemical observations.

VAN DUIJN, P.

doi: 10.1177/9.3.234pmid: 13888296

A cytochemical method based on the reaction of acrolein with tissue compounds followed by staining with the Schiff reagent is described. The reaction is completely blocked by acetylation.Filter paper spot tests of carbohydrates, nucleic acids and proteins revealed a high specificity for proteins. All proteins except the argininerich protamines were positive in the concentrations tested.Carbohydrates, as well as ribonucleic acid, were negative; deoxyribonucleic acid reacted weakly, probably as a result of a contamination. Tests with compounds of low molecular weight revealed that amino acids were positive, nucleic acid derivatives negative. Arguments are given that the double bond of acrolein reacts with SH, with aliphatic NH2 and NH groups, and with the imidazole group.The use of this method as a cytochemical stain in combination with Feulgen, thionine-SO2 staining is indicated, especially for chromosome studies. The method also seems applicable to paper and agar electrophoresis.

PUGH, DOREEN; WALKER, P. G.

doi: 10.1177/9.3.242pmid: 14489157

α-Naphthyl N-acetyl-β-glucosaminide has been prepared and used in a simultaneous azo-dye coupling method for the demonstration of N-acetyl-β-glucosaminidase activity in formalin fixed frozen sectionsThe effect of fixation on the activity of the enzyme has been invesitgated. In addition the effects of varying the substrate concentration, amount of diazonium salt and pH of the incubating medium are describedN-Acetylglucosaminonolactone, a specific inhibitor of N-acetyl-β-glucosaminidase has been used in experiments designed to test the specificity of the reaction.The relative activity of the enzyme in rat tissue homogenates has been measured and its distribution in rat tissues and human cartilage and synovial membrane is describedA satisfactory localization of N-acetyl-β-glucosaminidase is possible by the technique described. Factors affecting the histochemical localization are discussed in relation to the results obtained with other enzymes

WELCH, ROBERT M.; HANLY, E. W.

doi: 10.1177/9.3.251pmid: 14005976

Feulgen cytophotometry has been applied over a period of about two years to the analysis of the deoxyribonucleic acid content of 658 samples of spermatozoa from 275 Santa Gertrudis bulls of the King Ranch, most of which were under two years of age. A system of correction for smear to smear and slide to slide staining variation due to experimental error is presented that restricts the total maximum experimental error of a single determination to 15%, and to 10% in 80% of samples, when 18-25 sperm are measured per smear. Refinements of technique are described by which the maximum error can be reduced to 7% in 95% of samples and to 5% by increase in the number of sperm measured per sample smear.

MILCH, ROBERT AUSTIN; TOBIE, JOHN E.; ROBINSON, ROBERT A.; EVANS, CHARLES B.

doi: 10.1177/9.3.261pmid: 14473823

MANCINI, R. E.; VILAR, O.; DELLACHA, J. M.; DAVIDSON, O. W.; CASTRO, A.

doi: 10.1177/9.3.271pmid: 14469150

Several Armour thyrotrophin preparations were labeled with different amounts of a fluorescent dye, acid rhodamine B (Lissamine-Rhodamine B 200, Imperial Chemical Industries, Ltd.) following Chadwick's method with slight modifications. The native thyrotrophin lost from 12 to 34% of its biological activity after labeling, proved by Gilliland's method. A portion of the same labeled thyrotrophin was denaturated by heating in acetic acid medium. Different lots of rats were intravenously injected with native labeled thyrotrophin, denaturated labeled thyrotrophin and fluorescent dye solution respectively.It was observed: 1) Native labeled thyrotrophin declined rapidly in the circulation, though it remained longer than denaturated labeled thyrotrophin. 2) Fluorescence corresponding to native thyrotrophin was seen for a very short time in the thyroid gland and longer in the connective tissue of ocular structures, interstitium of skeletal muscles, perivisceral adipose tissue and mast cells. The Kupffer cells and other macrophages as well as the proximal tubule cells showed the longest accumulation. 3) Fluorescence corresponding to denatured labeled thyrotrophin was not observed in the thyroid gland; it was seen with low intensity in the connective and adipose tissues as well as in the mast cells. It was observed with higher intensity in the macrophages, convoluted tubule cells and it was seen to be eliminated through the epithelium of the small intestine. 4) Rats injected with acid rhodamine B solution eliminated the dye through the urinary and bile systems. In these animals fluorescence was seen only in the macrophages and proximal tubule cells, though in lesser amount compared with the animal injected with labeled hormone.

MANCINI, R. E.; VILAR, O.; STEIN, E.; FIORINI, H.

doi: 10.1177/9.3.278pmid: 14469152

A histochemical and radioautographic study of the connective tissue of rat skin was carried out from birth to maturity. The following observations were made:1) Early fibroblasts changed into typical adult connective tissue cells, fibrocytes, with gradual diminution of cytoplasmic nucleoproteins, periodic acid Schiff positive material, succinodehydrogenase, β-glucuronidase and leucyl-aminopeptidase.2) Lack of striking morphological or cytochemical changes of the mast cells in the different ages as compared with fibroblasts, but more intense enzymatic reactions with respect to succinodehydrogenase, β-glucuronidase, leucyl-aminopeptidase, alkaline and acid phosphatase, lipase esterase and cytochrome oxydase were found.3) Using adenine-C14 and methionine-S35, the localization of these substances was seen more in the fibroblasts and mast cells than in the intercellular ground substance; in the fibroblasts the uptake was higher in the embryonic and early postnatal connective tissues than in the adult.4) The embryonic and early postnatal connective tissues to synthesize sulphated polysaccharides trapped more S35 than the adult. S35 was seen first in the fibroblasts and then in the intercellular ground substance. The same results were observed in tissue cultures of fibroblasts. Mast cells incorporated and stored more S35 than fibroblasts, but there was no appreciable difference in their capacity for this at various ages, as there was in fibroblasts.

CHANG, JEFFREY P.; HORI, SAMUEL H.

doi: 10.1177/9.3.292pmid: N/A

A new technique of freeze-substitution, called "Section Freeze-Substitution," is described. Thin pieces of tissue are quick-frozen as for freeze-drying, and sectioned in a cryostat. The free sections are kept in dry ice-temperature absolute acetone overnight, then picked up with cover glasses, coated with celloidin if necessary, and air dried. The sections adherent to the cover glasses can be stained or incubated in substrate solution directly at room temperature (23-27°C).Finished slides can be made front fresh tissues[See figure in the PDF file]within 24 hours. With few exceptions, both oxidative and hydrolytic enzymes, soluble isotopes, and other chemical substances can be precisely demonstrated in a single substitution process. The cytology of the frozen-substituted tissue is excellent. No other histochemical tissue processing method preserves so much of a particular enzyme. The procedure is simple and the results are highly reproducible.

BURSTONE, MARVIN S.; WEISBURGER, ELIZABETH K.

doi: 10.1177/9.3.301pmid: 13875050

A histochemical technique for the diazotization of highly insoluble amines is described. The diazotization procedure is carried out in the presence of inert solvents, dimethylformamide or demethylsulfoxide.Most of the amines contain coordinating groups which facilitate chelation of the final dyestuff with metals, including copper and cobalt. The aforementioned coordinating groups include nitrogen, keto, hydroxyl, amino, carboxyl, and the azo grouping. Several dye chelates were found to be highly resistant to xylene and ethanol.In addition to the successful application of chelation techniques to azo-dye enzyme histochemistry, the first use of a metal containing diazonium salt is reported. The compound is diazotized p-(acetoxymercuri)-aniline. Diazotized 3-aminofluoranthene is highly stable in solution and is suitable for use in simultaneous coupling techniques.Use of complex diazotized amines possessing coordinating groups for subsequent chelation enhances chromogenicity and fineness of localization and may find application in electron microscopy.

TAKEUCHI, TADAO; GLENNER, GEORGE G.

doi: 10.1177/9.3.304pmid: 13919258

Polysaccharide was histochemically synthesized in muscle fibers and other tissue cells by using uridine diphosphate glucose as substrate. By revising the method previously described in light of new biochemical and histochemical findings more satisfactory optimal conditions were defined for the demonstration of the uridine diphosphate glucose-glycogen transferase enzyme.The histochemical properties of the reaction were very similar to those reported biochemically and this tended to verify the specificity of the reaction. Glucose-6-phosphate stimulated intensely the uridine diphosphate glucose-glycogen transferase reaction in an alkaline range.