PRESERVATION OF PHENYL-PHOSPHATASE ACTIVITY DURING PARAFFIN EMBEDDINGDALGAARD, JÖRGEN B.
doi: 10.1177/4.1.14pmid: 13295566
Model experiments with serum-gelatine pieces in which the effect of some basic histotechnical procedures on the phosphatase activity was studied, are reported. Following fixation and dehydration in alcohol, clearing in methyl-benzoate-celloidin, and embedding in paraffin at temperatures up to 53°C., the phosphomonoesterase activity was preserved, while embedding at higher temperatures caused increasing inactivation. The admixture of slight amounts of "soft paraffin" (m.p. 42°C.) to the ordinary 55° paraffin and embedding at 52-53° is advocated for phosphatase studies.
PROTEIN BINDING OF AZO DYES BY TISSUE HOMOGENATESRUTENBURG, ALEXANDER M.; SELIGMAN, ARNOLD M.
doi: 10.1177/4.1.17pmid: 13295567
In the colorimetric estimation of sulfatase activity in tissues based upon the conversion of 6-benzoyl-2-naphthol, liberated by enzymatic hydrolysis, to an azo dye by exposure to tetrazotized diorthoanisidine, a substantial fraction of the dye is adsorbed by tissue homogenate. This also occurs with protein fractions. The fraction of total dye adsorbed increases with protein or homogenate concentration and varies with the type of protein or homogenate used. The adsorption by protein conforms empirically to a general theoretic equation for the binding of azo dyes to protein. Azo dyes derived from 6-bromo- or 6-benzoyl-2-naphthol were adsorbed more extensively than the dyes derived from 1- or 2-naphthol.
A STRICTER INTERPRETATION OF THE FERRIC FERRICYANIDE REACTION WITH PARTICULAR REFERENCE TO THE DEMONSTRATION OF PROTEIN-BOUND SULPHYDRYL AND DI-SULPHIDE GROUPSADAMS, C. W. M.
doi: 10.1177/4.1.23pmid: 13295568
(1) Two distinct pigments (Prussian blue and Prussian green) are found in tissues after the ferric ferricyanide reaction; they have different chemical properties.(2) Only strong reduction causes rapid formation of Prussian blue from ferric ferricyanide solutions. Conversely Prussian green may be formed by weak reduction or possibly by absorption of traces of this pigment present in ferric ferricyanide solutions. Brown or yellow pigments in tissues may appear green in the presence of low concentrations of Prussian blue. Thus no strict interpretation of green colours can be made.(3) Sulphydryl groups, phenols, indoles, pyrroles, aromatic amines, uric acid and ascorbic acid reduced ferricyanide to Prussian blue. Certain oils, aliphatic amines, aldehydes and some inorganic reagents caused the production of Prussian green.(4) Up to 5 minutes immersion in the ferric ferricyanide solution is recommended for the demonstration of sulphydryl groups in trichloracetic-alcohol fixed tissues.(5) A technique is given for the demonstration of protein-bound disulphides, involving previous thioglycollate reduction followed by the demonstration of the formed sulphydryls by 1½ minutes immersion in the ferricyanide solution. This technique demonstrates protein containing at least 4 per cent of cystine (equivalent to 1 per cent of sulphur).
SPECIFICITY IN THE ALDEHYDE-COUPLING REACTIVITY OF NUCLEATED ERYTHROCYTESLESSLER, M. A.
doi: 10.1177/4.1.36pmid: 13295569
The nucleated erythrocytes of Amphiuma, Necturus, Rana pipiens, Rana catesbeiana, Siredon, and Gallus were exposed to a series of aldehyde-coupling reactions. The degree of coupling was indicated by subsequent blocking of Feulgen coloration measured visually and photometrically. Characteristic variation from species to species of the inhibition of Feulgen coloration was observed. These differences are interpreted as qualitative differences in the reactivity of the DNA complexes in the species studied.
PHOSPHORYLATION AS A HISTOCHEMICAL PROCEDURELANDING, BENJAMIN H.; HALL, HAZEL E.
doi: 10.1177/4.1.41pmid: 13295570
(1) The use of phosphoryl chloride (phosphorus oxychloride) in pyridine as a reagent for phosphorylation of tissue components is described.(2) Phosphorylation of many carbohydrate-containing substances is obtained by this method, including mucins, connective tissue (elastic tissue, reticulum, collagen, bone matrix), some glycolipids, glycogen and other tissue components.(3) Certain chemical entities that can be phosphorylated probably do not contain carbohydrate, but are lipids (either unsaturated or incompletely esterified), or proteins with free sulfhydryl, hydroxyl, or amino groups.(4) Phosphorylation reduces metachromasia of some tissue components (e.g. cartilage matrix), but enhances that of many others. The basophilia and metachromasia of phosphorylated components persist when staining is done in strongly acid solutions.(5) The significance of direct leukofuchsin positivity of many phosphorylated substances, and of relatively red staining of such substances with alum hematoxylin, and aldehyde-fuchsin, is not certain.
FACTORS INFLUENCING THE DETERMINATION OF THE INTRACELLULAR DISTRIBUTION OF ISOCITRIC DEHYDROGENASE IN RABBIT CEREBRAL CORTEXSHEPHERD, JAMES A.
doi: 10.1177/4.1.47pmid: 13295571
Washing the sediment or the mitochondrial fraction of rabbit cerebral cortex homogenates in the process of tissue fractionation increases its isocitric dehydrogenase activity thus giving anomalous recovery results. This increase on washing is not due to the removal of a soluble inhibitor of the dehydrogenase itself.Isocitric dehydrogenase activity is associated with the mitochondrial fraction and soluble fraction of rabbit cerebral cortex homogenates. It is unlikely that the mitochondrial or soluble activities have been derived from one another, or are due entirely to contamination of one fraction with another. The enzymes themselves from these sources have not been shown to be either the same or different enzymes. The activation of the particulate activity by freezing and thawing has been used as a roughly quantitative test for particulate bound activity. Mitochondrial bound enzyme may be activated by freezing and thawing, exposure to sonic vibrations, the shearing effect of Waring Blendor blades, and partially by washing. The latter may be explained on the basis of permeability factors aggravated by removal of isocitrate within the mitochondria by aconitase. Rabbit cerebral cortex mitochondria are not easily ruptured by homogenization in the Potter and Elvehjem homogenizer in sucrose suspension. The increase in specific activity of the nuclear fraction due to freezing and thawing is larger than that of the mitochondrial fraction, though the actual increase in activity is much smaller. The presence of a nuclear isocitric dehydrogenase has not been ruled out. A highly active isocitric dehydrogenase in soluble form has been prepared from rabbit cerebral cortex mitochondria.
A HISTOCHEMICAL SPECIFIC THIONINE-SO2 REAGENT AND ITS USE IN A BI-COLOR METHOD FOR DEOXYRIBONUCLEIC ACID AND PERIODIC ACID SCHIFF POSITIVE SUBSTANCESVAN DUIJN, P.
doi: 10.1177/4.1.55pmid: 13295572
A histochemical specific blue aldehyde reagent containing thionine and SO2, in a medium of tertiary butyl alcohol and water is described.The reagent, as far as visual observation goes, reacts in a constant high intensity with aldehyde locations over a period of at least five weeks. In artificial light however the shade of the dye-aldehyde combinations shows some variation.This unexplained phenomenon may point to a complicated reaction mechanism or may be related to the metachromatic qualities of the dyes.For this reason the use of the reagent in quantitation experiments still requires further study though it is more constant in shade and reaches a higher intensity in shorter times than previously described mixtures of thionine and SO2.When Feulgen hydrolyzed sections are stained with this reagent followed by the periodic acid Schiff procedure an image results of blue nuclei combined with the well known red extranuclear PAS pattern.With the exception of glycogen the PAS positive polysaccharides withstand the preceding Feulgen hydrolysis. By visual observation no indication of significant overlapping of colors was found.