A CHEMICAL AND HISTOCHEMICAL INVESTIGATION OF GLYCOGEN IN RAT LIVER AND PALATE FOLLOWING TREATMENT WITH VARIOUS FIXATIVES AND ETHYLENEDIAMINE TETRAACETIC ACIDTROTT, J. R.; GORENSTEIN, S. L.; PEIKOFF, M. D.
doi: 10.1177/10.3.245pmid: N/A
The loss of glycogen after fixation in acetic acid-alcohol-formalin as compared to immediate determination was found by chemical analysis to be from 5.2% to 13.8%. No appreciable difference could be seen histochemically.Chemical analysis of the glycogen content of liver fixed in acetic acid-alcohol-formalin and then treated in ethylenediamine tetracetic acid showed a total drop of 20.7 to 33.7%. Glycogen could still be adequately shown histochemically.Sections taken from liver fixed in 1% periodic acid in 10% formalin show greater amounts of glycogen, than those taken from acetic-acid-alcohol-formalin fixed tissue.After fixation in 1% periodic acid in 10% formalin and following treatment with ethylenediamine tetraacetic acid, very little glycogen could be demonstrated histochemically.In the palate, no glycogen could be demonstrated after fixation in acetic acid-alcohol-formalin, whereas quite heavy amounts could be seen in the prickle cell layers of the epithelium following fixation in 1% periodic acid in 10% formalin.Neither acetic acid-alcohol-formalin nor 1% periodic acid in 10% formalin are satisfactory fixatives if the tissue is to be further decalcified in ethylenediamine tetraacetic acid, if there are only small amounts of glycogen present.No adequate method of retaining glycogen in small amounts in tissue requiring decalcification can be suggested.
A PARAFFIN EMBEDDING TECHNIQUE FOR STUDIES EMPLOYING IMMUNOFLUORESCENCESAINTE-MARIE, GUY
doi: 10.1177/10.3.250pmid: N/A
A method is described for the fixation of blocks of tissue for use in studies employing immunofluorescence. This method consists of fixing thin blocks in 95% ethanol and carrying out the subsequent dehydration and clearing at refrigerator temperatures (4°C). Thereafter, embedding in paraffin and sectioning by the standard microtomy is easy.This method results in preparations which are histologically more precise in the localization of antigen or antibody than preparations of frozen tissues; and with rabbit antibody and bovine serum albumin, the sensitivity of detection is enhanced. Bovine serum albumin can be found for longer periods after injection than is possible with frozen sections.Other antigens for which this procedure has proved satisfactory are bovine gamma globulin, horse ferritin, influenza A virus, diphtheria and tetanus toxoids. Hen's ovalbumin deteriorated.New antigens or new antibodies should be tested before being committed to this method.
QUANTITATIVE HISTOCHEMISTRY OF THE KIDNEY. II. ENZYMATIC ACTIVITIES IN GLOMERULI AND PROXIMAL TUBULES IN AMINONUCLEOSIDE NEPHROSIS IN RATSKISSANE, JOHN M.; HOFF, EUGENE
doi: 10.1177/10.3.259pmid: N/A
Quantitative determinations of enzymatic activities in glomeruli and in two segments of proximal tubules at intervals in the development of aminonucleoside nephrosis in rats suggest that several mechanisms are at play in this experimental disease. Increased glucose-6-phosphate dehydrogenase in glomeruli, first demonstrated by Dubach and Recant, and increased 6-phosphogluconic dehydrogenase in glomeruli were the earliest enzymatic alterations which were demonstrated and constitute evidence that the glomerulus is the renal structure primarily deranged in this form of experimental nephrosis. The exact biochemical nature of the glomerular lesions remains obscure. Decreased tubular alkaline phosphatase was correlated with tubular dilatation from obstruction by casts. Decreases were found in tubular activities of malic, glutamic, and isocitric dehydrogenases, and of glycylphenylalanine dipeptidase; similar changes in these activities were observed in experimental proteinuria.In aminonucleoside-treated animals, slight increases in glomerular malic, lactic, and glutamic dehydrogenases occurred after 11 to 15 days, appreciably later than the increases in enzymes of the hexose monophosphate pathway.Increased tubular activity of acid phosphatase may be a component of non-specific cellular degeneration.
IMPROVEMENTS IN THE COATING TECHNIQUE OF RADIOAUTOGRAPHYKOPRIWA, BEATRIX MARKUS; LEBLOND, C. P.
doi: 10.1177/10.3.269pmid: N/A
The 1958 coating technique of Messier and Leblond for radioautography—the most recent modification of the liquid emulsion method—was investigated in the hope of eliminating artefacts and discovering the optimum conditions for reliable quantitative radioautography.From a review of the properties of the various bulk emulsions available, it is concluded that NTB2 bulk emulsion is the most suitable. The proper handling of the emulsion (transportation, storage and testing operations) is described, as well as the details of an improved coating techniqe. Comparison is made of this method with the other most widely used, the strippingfilm technique.The main features of the improved coating technique are as follows:1. Various fixatives may be used for radioautography. Zenker and presumably other mercury-containing fixatives are unreliable, but can be used after complete removal of mercury salts.2. Dipping the sections into celloidin prevents the infiltration of the tissue by emulsion, but is otherwise unnecessary and may be omitted for routine work.3. Emulsion coating is done by dipping sections in a bubble-free emulsion at 40°C.4. Drying of the coated preparations is best accomplished by keeping them vertical over moistened tissue paper at 28°C and 80% relative humidity.5. Exposure is done in a dry atmosphere at 4°C.6. Processing is carried out at 17-18°C, as even a slight rise in temperature increases the background fog and decreases the efficiency of the emulsion. The optimal duration of development is 2 minutes for D-72 (Dektol), 6 minutes for D-l70 (Dolmi) and 3 minutes for D-178. For fixation, best results are obtained using acid fixer with hardener (or 24% hypo) for 3 minutes.7. Mounting is done by successive immersions in an alcohol-cedar oil mixture, a xylene-balsam mixture, and balsam. Malinol may replace balsam.The improved coating technique arising from these observations was tested for its quantitative response, measured by grain counts. The suitability of the technique for quantitative radioautography is demonstrated by the fact that the reaction intensity is proportional to exposure up to at least 80 days with hematoxylin-cosin stained sections, and 1 year with unstained sections. The background increases slowly with time, but the increase is more rapid on slides with C14-labelled sections than on those labelled with H3.In conclusion, the improved coating technique using the NTB2 emulsion is now quite satisfactory. The next major improvement would be for the industry to provide emulsions with a standardized sensitivity and a constant, low level of fog. Nevertheless, in its present state, it is a convenient method permitting the preparation of large numbers of high quality radioautographs and is well suited to the grain counting required for quantitative radioautography.
HISTOCHEMISTRY AND ELECTRON MICROSCOPY OF MITOCHONDRIAL ADENOSINETRIPHOSPHATASELAZARUS, SYDNEY S.; BARDEN, HERBERT
doi: 10.1177/10.3.285pmid: N/A
A study of the requirements for histochemical demonstration of mitochondrial adenosinetriphosphatase demonstrated that fixation of cryostat sections for 15 minutes in cold 0.05 M manganese chloride in 3% formol saturated with CaCO3 (pH 6.9) gave best results. By this means the enzyme could be demonstrated with both the calcium and lead incubation media. The adenosinetriphosphatase activity was demonstrated ultramicroscopically and was found to be compartmentalized in the intereristal matrix.The histochemically demonstrated enzyme showed responses to inhibitors and activators characteristic of adenosinetriphosphatase of damaged mitochondria obtained by ultracentrifugation.
A METHOD FOR THE HISTOCHEMICAL DEMONSTRATION OF DISACCHARIDASE ACTIVITIES: APPLICATION TO INVERTASE AND TREHALASE IN SOME ANIMAL TISSUESDAHLQVIST, ARNE; BRUN, ARNE
doi: 10.1177/10.3.294pmid: N/A
1) A histochemical method for the demonstration and localization of disaccharidase activity using various disaccharides as the substrate is described. Frozen sections are incubated in a solution containing substrate plus a coupled oxidation-reduction system of glucose oxidase, phenazine methosulfate and a tetrazolium salt. Where disccharidase activity is present, the substrate is hydrolyzed with the liberation of glucose, and a deep blue insoluble formazan is formed.2) This method has been used for the demonstration of invertase and trehalase in rat tissue sections. The small intestine, colon, stomach and kidney were investigated.3) All the epithelial cells of the small intestinal mucosa, in the villi, crypts and glands of Brunner, contain invertase and trehalase. These enzymes appear to be localized to small rounded granules which are scattered throughout the cytoplasm of the cells. The activity is especially high in the perinuclear part of the epithelial cells, but no concentration of the granules is to be seen in "brush border" region.4) The stomach and the kidney also contain weak invertase activity which is present in granules localized to small distinct cell groups. No trehalase activity could be demonstrated in these organs. The colon contains essentially no invertase or trehalase.
NEW OBSERVATIONS ON DISCREPANCIES IN THE HISTOCHEMICAL LOCALIZATION OF LEUCINE AMINOPEPTIDASENACHLAS, MARVIN M.; FRIEDMAN, MELVIN M.; SELIGMAN, ARNOLD M.
doi: 10.1177/10.3.315pmid: N/A
Differences in the localization of leucine aminopeptidase in the cortex of the rat kidney have been reported with two methods employing the same substrate. Test tube and tissue experiments were carried out in an effort to explain the disparity. Substrate concentration, pH, buffer, the use of an activator, duration of incubation and temperature of incubation were found to have insignificant effects. Four important findings were (1) leucine aminopeptidase activity of the inner cortex of the rat kidney assayed two to three times greater than that of the outer cortex, (2) this difference was confirmed by histochemical demonstration with both methods, (3) fast blue B inhibited the enzyme more than fast garnet GBC and fast Corinth V, and (4) fast blue B coupled faster than the other diazonium salts.