A STUDY OF OSMIUM TETROXIDE FIXATIONBURKL, W.; SCHIECHL, H.
doi: 10.1177/16.3.157pmid: 4870843
The specific conditions of osmium fixation have been studied on gelatin gels and bovine pancreas. Protein (gelatin)-bound osmium can be quantitated by a modification of the Majumdar and Sen Gupta test, free and tissue (pancreas)-bound osmium by a modified Bahr test. Using these two methods, the depth of OsO4 penetration after increasing the times of fixation, the osmium concentration gradients and the total concentration of free and bound osmium at given spots within gelatin and tissue blocks have been determined. Warburg type experiments on suspended yeast cells and leukocytes demonstrate that 1% OsO4 stops cell respiration completely only after 40-min incubation. Formalin, 1%, is a more effective respiratory inhibitor.
CYTOCHEMISTRY OF 5-HYDROXYTRYPTAMINE AT THE ELECTRON MICROSCOPE LEVEL I. STUDY OF THE SPECIFICITY OF THE REACTION IN ISOLATED BLOOD PLATELETSETCHEVERRY, GUILLERMO JAIM; ZIEHER, LUIS MARIA
doi: 10.1177/16.3.162pmid: 5652099
Blood platelets obtained from normal rabbits and those isolated from reserpine-treated animals and subsequently incubated in vitro with 5-hydroxytryptamine, norepinephrine and histamine were assayed for amine content or processed for examination under the electron microscope. With the glutaraldehyde-dichromate reaction for unsubstituted catechol- and indoleamines, reactive granules were observed in normal platelets. Formaldehyde fixation prior to the glutaraldehyde-dichromate reaction resulted in a similar image under the electron microscope. In platelets obtained from animals treated with reserpine a decrease of the amine content with a corresponding reduction in the number of dense granules was observed. Following incubation with 5-hydroxytryptamine the concentration of the amine increased markedly and the number of dense granules that reacted with both techniques became practically normal. In norepinephrine-incubated platelets dense granules were demonstrated with the glutaraldehyde-dichromate reaction, but no reactive products were observed using prefixation with formaldehyde. Histamine was also incorporated into depleted platelets but gave no reaction. It is concluded that prefixation with formaldehyde renders negative the reaction with catecholamines, leaving unaffected indoleamine-reactive sites. The previous assumption that the dense granules contain 5-hydroxytryptamine has been confirmed by such a cytochemical approach. The possibility that these organelles constitute a common storage site for different amines is discussed.
CYTOCHEMICAL DEMONSTRATION OF ORNITHINE CARBAMOYLTRANSFERASE ACTIVITY IN LIVER MITOCHONDRIA OF RAT AND MOUSEMIZUTANI, AKIRA
doi: 10.1177/16.3.172pmid: 4172322
In order to demonstrate ornithine carbamoyltransferase activity cytochemically, thin slices of liver and kidney of rat and mouse were fixed in cold acetone or formol-calcium, and incubated in a medium containing l-ornithine, carbamoyl phosphate, Tris-maleate buffer (pH 7.2), lead nitrate and sucrose. The specific reaction product occurred in the mitochondria of the hepatocytes only, and not in other cells of the liver or kidney. The specificity of the reaction was supported by the following observations. (1) The mitochondrial reaction was not obtained in sections incubated in a medium from which ornithine was omitted. (2) Other amino acids gave no reaction. (3) p-Chloromercuribenzoate suppressed the reaction. (4) The hepatocytes of chick (uricotelic) did not give the reaction. A nonspecific reaction in lysosomes and brush borders is caused probably by acid and alkaline phosphatase activities.
THE EFFECTS OF LEAD AND FIXATIVES ON ACTIVITY OF GLUTAMIC OXALACETIC TRANSAMINASELEE, SIN HANG; TORACK, RICHARD M.
doi: 10.1177/16.3.181pmid: 5652100
Lead oxalacetate, a primary reaction product precipitate for the visualization of the activity of glutamic oxalacetic transaminase in tissue sections, is stable at a slightly alkaline pH. At concentrations which are used for tissue fixation a slight inhibitory effect on glutamic oxalacetic transaminase activity is produced by acetone while a glutaraldehyde-formaldehyde mixture results in marked reduction of activity. The concentration of lead which is used to form the primary reaction product does not significantly inhibit this enzyme activity.
HISTOCHEMICAL DIFFERENTIATION BETWEEN TYROSINE AND CHLOROGENIC ACID IN PLANT TISSUES I. NITROUS ACID REACTIONS AND METAL CHELATION OF NITROSOTYROSINEREEVE, R. M.
doi: 10.1177/16.3.191pmid: 5652101
Copper chelation of nitrosotyrosine has been found useful for histochemical localization for tyrosine in thick, fresh sections of large celled plant tissues. The nitrous acid reaction for ortho-dihydroxyphenolics also has been found useful for localization of chlorogenic acid, caffeic acid and dihydroxyphenylalanine in plant tissues. Application of these tests separately to serially adjacent sections demonstrated the distribution of tyrosine and chlorogenic acid in different plant tissues. Tests tube reactions on known substances verified specificity and also demonstrated that the presence of other amino acids and phenolics did not interfere with the positive test for tyrosine. The color reactions are sufficiently intense for stereoscopic microscopy and tested sections may be measured photometrically. Further adaptability of the nitrosotyrosine-metal chelate reaction to procedures for ultrastructural localization is suggested.
PURIFICATION OF GLUTARALDEHYDE ITS SIGNIFICANCE FOR PRESERVATION OF ACID PHOSPHATASE ACTIVITYFAHIMI, H. DARIUSH; DROCHMANS, PIERRE; POPOWSKI, A.
doi: 10.1177/16.3.199pmid: 4870844
The inhibition of acid phosphatase activity in rat liver homogenates after fixation in different lots of commercial glutaraldehyde is determined and compared with the inhibition following fixation with a distilled product. It is shown that commercial glutaraldehydes inhibit more of the enzyme activity than the distilled product. The acidic products of oxidation of glutaraldehyde do not increase the inhibition of the enzymatic activity. The presence of high concentration of inorganic phosphates in different lots of commercial glutaraldehyde, as presented here, suggests that probably such impurities may be responsible for increased inhibition of phosphatase activity noted after fixation in commercial glutaraldehydes.