An alcohol-soluble Schiff's reagent: a histochemical application of the complex between Schiff's reagent and phosphotungstic acid.

A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent used as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.     

A mechanistic study of the histochemical reactions between aldehydes and Basic Fuchsin in acid alcohol used as a simplified substitute for Schiff's reagent

Synopsis For the identification of polysaccharides after periodic acid oxidation or of DNA after acid hydrolysis, a solution of 0.5% w/v Basic Fuchsin in acid alcohol (water-ethanol-concentrated hydrochloric acid 80:20:1 by volume) may be used instead of Schiff's reagent. Sections are stained in the Fuchsin solution for 20 min, after which the unreacted dye is washed off with ethanol. Except for its yellower colour the Fuchsin staining is almost indistinguishable from Schiff's reagent staining.Histochemical blocking studies indicated that the Fuchsin stain, like Schiff's reagent, reacts with aldehyde groups or subsequent oxidation products. The results of studies of model systems (cellulose film oxidized by periodic acid and also of aqueous formaldehyde solution) in which infra-red spectroscopy and, where appropriate, chromatography were used are consistent with the initial coloured products being azomethines which may react further to produce coloured secondary amine derivatives.     

Fluorescent-labelled aprotinin: A new reagent for the histochemical detection of acid mucosubstances

Summary Aprotinin (Trasylol®), a polypeptide protease inhibitor from bovine organs, has been labelled with fluorescein isothiocyanate to produce a fluorecent conjugate (FLA). The conjugate has been applied to paraffin sections of rat tissues and has been shown to act as a specific fluorochrome for acidic mucosubstances. There is reason to believe that FLA stains carbohydrates which owe their acidity to the presence of carboxyl groups. The histochemical findings are described and discussed and it is also suggested that attachment of aprotinin to extracellular polysaccharides in vivo may limit the therapeutic usefulness of the drug.     

The histochemical application of dansylhydrazine as a fluorescent labeling reagent for sialic acid in cellular glycoconjugates.

A new method for the fluorescent staining of stalic acid-containing glycoconjugates in fixed tissues is described. The procedure uses mild periodate oxidation, followed by condensation with dansylhydrazine and reduction of the hydrazones to hydrazines. The specificity of the reaction for sialic acid is tested on model glycoconjugates. The procedure gives superior resolution in comparison to the standard periodate Schiff procedure for cellular carbohydrates.     

Sequential staining of DNA-aldehyde with Schiff's reagent and acriflavine-SO2.

Acid hydrolysed DNA of rat liver was stained with Schiff's reagent at pHs 1.7 or 3.0 followed by staining with acriflavine-SO2 at pH 2.0 as well as with acriflavine-SO2 followed by Schiff's reagent at pH 1.7 or 3.0. Nuclei stained with Schiff's reagent at pH 1.7 were brown-yellow and an analysis of their absorption characteristics revealed two peaks--one at 470 nm and the other at 570 nm. Although nuclei stained with Schiff's reagent at pH 3.0 followed by acriflavine-SO2 were deep magenta in colour, they also showed similar peaks of maximum absorption. Identical peaks were also seen when the sequence of staining was reversed. It is suggested that in the conventional Feulgen-type reactions only some of the DNA-aldehyde molecules are stained; the remaining molecules can be stained by sequential application of another Schiff or Schiff-type reagent such as acriflavine-SO2. The possible mechanism of staining in these cases has been discussed.     

On the specificity of alcoholic acidic silver nitrate reagent for the histochemical localization of ascorbic acid

Summary The specificity of the alcoholic acidic silver nitrate staining method for the histochemical localization of ascorbic acid was reappraised. It was found that the method is by and large better suited for the localization of ascorbic acid in both animal and plant tissues due to its greater specificity, which is ensured by employing reagent made in carbon dioxide saturated glass distilled water as well as by carrying out the reaction at a low temperature (0–4° C) and at a pH of 2–2.5.Paper presented at the 65th Session, Indian Science Congress, Ahmedabad, Jan. 3–7, 1978     

An automated device for the preparation of complex reagent mixtures

A microcomputer-controlled device was built that automatically prepares small volumes of mixtures of up to eight reagents. The operation of the system is fast, flexible, and reliable, thus making possible the routine use of experimental protocols that require large numbers of small volume reagent samples, each having a different composition. In particular, the software we developed for this device handles the preparation of three-antibody staining solutions to be used in triple labeling immunofluorescent flow cytometry experiments that involve only two fluorochromes. In this role, the device is known as an “Immunofluorescence Tomograph.”     

On the specificity of alcoholic acidic silver nitrate reagent for the histochemical localization of ascorbic acid. A reappraisal.

The specificity of the alcoholic acidic silver nitrate staining method for the histochemical localization of ascorbic acid was reappraised. It was found that the method is by and large better suited for the localization of ascorbic acid in both animal and plant tissues due to its greater specificity, which is ensured by employing reagent made in carbon dioxide saturated glass distilled water as well as by carrying out the reaction at a low temperature (0-4 degrees C) and at a pH of 2-2.5.     

Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid.

The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0-4 degrees C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

On the specificity of the alcoholic,acidic silver nitrate reagent for the histochemical localization of ascorbic acid

Summary The defects besetting the histochemical localization of ascorbic acid were removed in the modified method described here by the simultaneous fixation of the experimental material and its reaction with silver nitrate by the use of alcoholic, acidic silver nitrate reagent in the dark at 0–3°C for 24 hours or longer at pH 2–2.5.The fixatives like acetic acid and alcohol of the reagent ensure quick penetration of AgNO₃ for fixation of ascorbic acid in situ before sectioning. It has been experimentally established that none of the other reductants react with AgNO₃ at the pH and the temperature mentioned.The sections were devitaminized by treatment with 6–10% formaline for 3–4 hours to serve as a control.