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S R Silveira

Publications and source records attributed to S R Silveira.

5 recordsLinked to original sources

Catalases and peroxidases histochemical detection; techniques suitable to discriminate these enzymes.

By using the benzidine reaction, on filter paper strips loaded with catalases, peroxidases, porphyrins, haemic iron compounds and iron salts, it was possible to establish 2 histochemical techniques able to detect and discriminate catalases and peroxidases. Spot test analytical studies show that only peroxidases oxidize benzidine in presence of a 0.0015 M H2O2 final concentration into the incubation medium. If a 0.0035 M H2O2 final concentration is used both peroxidases and haemic iron were able to oxidize benzidine. At a 0.01 M H2O2 final concentration the oxidative property of catalases become apparent and therefore at this H2O2 concentration either peroxidases or haemic iron, as well as catalases could be detected. By increasing the H2O2 concentration into the incubation medium, when a 4M concentration was chosen to detect histochemically catalases without any peroxidases interference. Using 0.0015 M and 4 M H2O2 final concentrations into the incubation medium it is possible to discriminate histochemically catalases and peroxidases. Several inhibitors of catalases and peroxidases were used as an attempt to try a specific inhibition of only one of these enzymes. It was demonstrated that the use of inhibitors does not help the histochemical discrimination between catalases and peroxidases.

Adrenal Glands

Histochemical technique to detect choline-containing lipids.

An almost specific and highly sensitive technique to detect histochemically choline-containing lipids is proposed. This technique is based on the effect of Hg++ and phosphomolybdic ion on the choline, that yields insoluble complexes able to react with diphenylcarbazide producing a deep blue or violet stain. The specificity and the sensitivity of this technique was investigated on filter paper strips loaded with choline-containing lipids, choline-free lipids, steroids, vitamins, proteins or choline hydrochloride. The results show that the choline-containing substances react positively. The choline-free substances react negatively except calciferol and vitamin A that show weakly positive results. This technique used on tissue sections appears very suitable to histochemical purposes.

Adrenal Glands

Immunochemical specificity of a benzidine technique proposed to catalases histochemical detection.

As an attempt to test the specificity of an histochemical technique proposed to detect catalases, an investigation was carried out by immunochemical techniques. Purified catalases were used after analysed immunochemically by double immune diffusion test and immunoelectrophoretic technique. These pure catalases induced, after injecting into guinea-pigs, anti-serums that react specifically with catalase and does not give any cross reaction with peroxidases and haemic iron containing compounds. By the direct and indirect immuno-fluorescence techniques it was shown an intense catalase reactivity inside the cytoplasm of adrenal cortex and hepatic cells, that appears as a granular pattern. These results are very similar to those provided by the histochemical technique, either concerning to the reactive cells or to the granular pattern of the positive reaction. In such instances, the immunochemical results suggest the specificity of the histochemical reaction. This specificity is confirmed by the previous treatment of tissue sections by catalases anti-serum. After this treatment either the immunochemical or the histochemical technique to detect catalases provide negative results on cells that before the treatment were strongly reactive.

Adrenal Cortex

An analysis on the specificity of the histochemical techniques already proposed to detect catalases and peroxidases.

The spot test carried on filter paper strips appears as a very suitable technique to investigate the reactivity of catalases, peroxidases, porphyrins (bilirubin and protoporphyrin), metalloporphyrins (haemoglobin, haemin, haematin, chlorophyll and cyanocobalamin), ferric and ferrous salts. By using this technique the specificity of the already proposed techniques admitted as appropriate to detect histochemically peroxidases and catalases was investigated. The results shown from the already proposed techniques to detect peroxidases only the alpha-naphthol reaction is somewhat specific for this enzyme, if the results were taken immediately. However, if the results were taken after 24 h, the reaction loose all specificity. The other techniques proposed to detect peroxidase are not specific, either concerning the discrimination between catalases and peroxidases activity or regarding the possibility to differenciate an enzymic from a catalytic activity provided by haemic iron containing compounds and sometimes by iron salts. Our histochemical technique already proposed as suitable to detect catalases seams to be specific, since peroxidases do not react positively. By replacing benzidine for some others hydrogen donors the peroxidases histochemical techniques remain not specific and are unable to discriminate this enzyme from catalases. Porphyrins (protoporphyrin and bilirubin), magnesium and cobalt containing metalloporhyrins (chlorophyll and cyanocobalamin) do not produce oxidation of any hydrogen donors used. Iron salts are also able to give positive results with some techniques already proposed as suitable for peroxidases and catalases detection.

Animals