Shelf-life of citric acid-toluidine blue O-SO2 and its influence on Feulgen staining.

The paper reports on the preparation of a dye-SO2 reagent employing a Schiff-type dye, toluidine blue O. The method is to replace 5 ml of N HCl per 100 ml of the dye solution by citric acid. The usual potassium metabisulphite is then added. The pH of this new modified dye-SO2 reagent is 2.5 as against 1.6 for the hydrochloric acid-toluidine blue O-SO2. The shelf-life of this newly developed dye-SO2 reagent is four weeks with appreciable reduction of staining intensity after this period as compared with that of a freshly prepared dye-reagent with N HCl. A possible interpretation for the observed phenomenon has been suggested.     

Use of tris-buffer-fortified thionine-SO2 for detection of DNA-aldehyde following Feulgen procedure

This communication presents informations on the use of tris-buffer along with N HCl and potassium metabisulphite for the preparation of thionine-SO2 in staining DNA-aldehyde molecules of acid hydrolysed mammalian liver sections. It has been found that thionine, containing tris-buffer, N HCl and potassium metabisulphite, stains DNA-aldehyde molecules with better result than is possible with the control dye-SO2 reagent that does not contain this buffer. The absorption spectra of nuclei stained with this dye-reagent prepared with tris-buffer have also been presented. Further, it has been found that nuclei stained with the freshly prepared dye-SO2 reagent is bluish-violet, whereas those stained with an old dye-reagent is sky blue in colour. The reason for the slightly enhanced nuclear colouration with the experimental dye-reagent over the control has been considered to be due to slightly increased pH in the former as compared with that of the latter. The mechanism of staining with thionine-SO2 has been considered to be of Feulgen type.     

Hydrochloric acid-sodium thiosulphate-Schiff reagent and its cytochemical properties.

Cytochemical properties of modified Schiff reagent, prepared with 5 ml of N HCl and 1.0 g of sodium thiosulphate, the latter replacing potassium metabisulphite, have been presented in this communication. It has been seen that such a modified Schiff reagent has a slightly elevated pH as compared with that of the conventional Schiff reagent. It has also been found that this modified Schiff reagent has a much longer shelf-life and that the staining intensity produced by this dye-reagent is more than those of the usual Schiff reagent prepared with N HCl and potassium metabisulphite (potassium pyrosulphite, K2S2O5). Possible mechanism as to why the staining intensity of the nuclei is more with this modified Schiff reagent as well as the reason as to why its shelf-life is increased compared with those of the conventional Schiff reagent have been discussed.     

Influence of UV rays on Feulgen-type staining with azure A-SO2 prepared with normal hydrochloric acid and sodium thiosulphate

This communication presents a new method for the preparation of azure A-SO2 for use in Feulgen procedure. The salient feature of this method lies in the fact that azure A-SO2 can be decolourised with normal hydrochloric acid and sodium thiosulphate. The pH of this dye reagent is 2.3 and it is of water colour after filtration. The pH of this dye-reagent is raised to 4.0 with an aqueous solution of sodium hydroxide. Nuclear colouration with this newly developed dye-reagent on acid-hydrolysed DNA of tissue sections becomes fairly satisfactory under the usual laboratory conditions. Staining with this dye-reagent under exposure to UV ray is, however, vastly improved within 5 minutes as compared with the control. Stained sections do withstand treatment in SO2 water without exhibiting any leaching of the dye from the nuclei. Possible mode of action of UV rays in increasing the intensity of staining as well as the speed of reaction has been suggested.     

Preparation of Schiff-type dye-SO2 reagents with phosphoric acid and their cytochemical properties

A new method employing phosphoric acid in place of hydrochloric acid for the preparation of azure A-SO2 and toluidine blue O-SO2 has been described in this communication. It has been found that o-phosphoric acid in a weak concentration can be a suitable substitute for hydrochloric acid in the preparation of these dye-SO2 reagents. These dye-reagents produce excellent blue nuclei in human uterine tumour. Toluidine blue O-SO2 fortified with an amino aicd, glycine also produces excellent blue chromosomes in the squash preparations of grasshopper testes fixed in acetic acid-alcohol. The shelf-life of these two dye-reagents has also been presented herein.     

Azure B staining of DNA-aldehyde molecules of acid-hydrolysed tissue sections

This communication presents a method for the preparation of azure B-SO2 with trichloracetic acid (TCA) and potassium metabisulphite for in situ demonstration of DNA-aldehyde molecules following acid hydrolysis of tissue sections. The shelf-life of such a dye-reagent is slightly more than that of the control, prepared with N HCl and potassium metabisulphite. The slightly increased shelf-life of the experimental dye-reagent has been considered to be due to a somewhat higher pH as compared with that of the control. The in situ absorption characteristics of nuclei stained for DNA-aldehyde molecules with either an aqueous solution of azure B or with TCA-azure B-SO2 show peak-absorption at 600 nm in both cases. This phenomenon has been interpreted as due to the fact that azure B does not contain any primary amino group in its molecules and, therefore, the mode of binding of DNA-aldehydes with this dye is different from that with dyes that contain primary amino group. The implications of some of the findings have been discussed.     

Feulgen type staining with Hoffmann's violet-SO2 under exposure to UV rays.

The paper contains an account of the use of Hoffmann's violet-SO2 under exposure to UV rays during staining acid-hydrolysed DNA of mammalian tissue nuclei. Preparations stained with Hoffmann's violet-SO2 without exposure to UV rays reveal extremely pale violet nuclei but when stained under the influence of UV rays show a considerably faster reaction resulting in a very much deeper staining of the nuclei. Sections after staining with this dye-reagent require n-butanol as differentiating reagent. Possible interpretation for the increase in staining ability of this dye-reagent under exposure to UV rays has been elucidated and the reason for considering the reaction as Feulgen type has been discussed.     

Staining of DNA-phosphate groups and DNA-aldehyde molecules with dyes of the azine group

The investigation reports on the use of safranine-SO2 and phenosafranine-SO2, prepared with N HCl or oxalic acid plus potassium metabisulphite, for staining rat liver sections following Feulgen procedure. It has been found that optimum staining of DNA-aldehyde molecules is possible with safranine-SO2 and phenosafranine-SO2, prepared with N HCl and potassium metabisulphite, upto a duration of one week after the preparation of the dye-reagents. Thereafter, staining intensity of the nuclei produced by the dye-reagents is gradually diminished. Staining of acid-hydrolysed sections is also possible with aqueous solutions of these dyes. Moreover, DNA-phosphate groups can also be stained with aqueous solutions of these dyes after selective extraction of RNA with cold phosphoric acid. The in situ absorption spectra of nuclei, stained for DNA-aldehyde molecules with safranine-SO2, phenosafranine-SO2 and aqueous solutions of these dyes, have been presented in this paper. Also presented herein are absorption data of nuclei stained with these dyes after selective extraction of RNA. It has been found that absorption-peaks of nuclei stained differently are different from one another. The implications of these findings have been discussed.     

Propionic acid-metabisulphite-Schiff reagent for quick Feulgen staining

This communication presents a method for the preparation of Schiff reagent in which N hydrochloric acid has been replaced by a low concentration of propionic acid. The result of using such a Schiff reagent indicates that the dye-reagent thus prepared is extrafast in action on acid-hydrolysed mammalian tissue sections. The intensity of nuclear colouration is also increased considerably, since the pH of the dye-reagent is 6.0. It is, therefore advocated that this newly developed Schiff reagent be used at 5 degrees C.     

In situ demonstration of DNA with Schiff-type dyes prepared with meta-phosphoric or tartaric acid

A new method for the preparation of azure A-SO2 and safranine-SO2 For use in Feulgen procedure has been described herein. The method involves the use of m-phosphoric acid or tartaric acid in place of N HCl in the preparation of these eye-reagents which exhibit enhanced pH producing increased staining intensity of the nuclei as compared with those of the controls, prepared with N HCl. Possible explanation for the increased staining intensity as well as the reason for the shorter shelf-life of these eye-reagents have been offered.     

Combining the gold chloride and toluidine blue stains to investigate chick embryo neural tissue

Toluidine blue and gold chloride stains are both well‐known for staining nervous tissue. Toluidine blue is a general method to stain neurons and glia, used because of the speed of action. Gold sublimate is a delicate procedure that requires extreme purity of reagents and materials, and it stains astrocytes and neurons. The aim of this study was to combine these two special staining methods for embryonic neural tissue and to obtain a repeatable and easily manageable new staining method with improved overall visualization of neural tissue. Fertilized Broiler hatching eggs were incubated at 36°C and on day 8 prepared for histology. Sections were cut at 7 μm thickness and slides were stained for 30 s using a 0.1% Toluidine blue solution. These slides were rinsed with dH₂O followed by placing them in gold sublimate for 4 h at room temperature in the dark. Rinsing with dH₂O and transferal to a 5% sodium thiosulfate solution was done, followed by another rinsing and mounting of slides. Results showed that the combination of the two methods offers a fast, reliable method with which chick embryonic neural tissue can be evaluated.     

Increased shelf-life and feulgen staining intensity of a modified trichloracetic acid-Schiff reagent.

Two modified formulae for the preparation of trichloracetic acid (TCA)-Schiff reagent have been presented in this communication. The modifications involve addition of 250 mg or 500 mg of trichloracetic acid per 100 ml of a 0.5% aqueous solution of basic fuchsin in place of NTCA which means 1.63 g of TCA per 10 ml of distilled water to be added to 100 ml of the dye solution. The use of TCA-Schiff reagents prepared by the modified method reveals a considerably faster Feulgen staining, a more intense colouration of the nuclei and a longer shelf-life of the dye-SO2 reagents as compared with the standard TCA-Schiff of Bloch and Godman. Possible interpretations for these phenomena have been presented.     

Acridine orange--its use in the specific staining of DNA in mammalian tissue sections

This paper reports on a new method for the use of acridine orange (AO) in an aqueous solution at pH 4.5 for staining DNA of rat tissue sections from which RNA has been extracted selectively with cold phosphoric acid. Not only this, AO can also be used as dye-SO2 reagent, prepared with NHCl and potassium metabisulphite, for staining DNA-aldehyde molecules of acid-hydrolysed tissue sections. AO samples, manufactured by the National Aniline Division as well as by G. T. Gurr have been used with equal success. Studies of stained sections under light microscope reveal the presence of specifically stained yellowish-orange nuclei. Those sections under fluorescent microscope with proper exciter and barrier filters reveal nuclei of maroon colour. The in situ absorption spectra of nuclei stained with AO-SO2 following acid-hydrolysis of tissue sections as well as those of nuclei stained with an aqueous solution of the dye following extraction of RNA have been presented herein. The mode of binding in the former case has been considered to be due to binding of the teritary amino group of the dye molecules with the DNA-aldehyde molecules and in the latter case to be due to electrostatic binding between the positively charged dye molecules with negatively charged phosphate groups of DNA. Implications of all these findings have been discussed.     

Studies in fluorescence histochemistry: II. The demonstration of periodate-reactive mucosubstances with pseudo-Schiff reagents*

According to Kasten, periodate-oxidized mucosubstances which have been stained with his fluorescent Schiff-type reagents (solutions of basic fluorescent dyes saturated with sulphur-dioxide) emit a specific fluorescence in sections of fixed tissue. In my experience it is sometimes difficult to observe this fluorescence because it is obscured by the similarly-coloured fluorescence emitted by proteins and nuclei. This and other difficulties, both theoretical and practical, can be mostly overcome by methylating tissue sections beforehand with a methanolic solution of thionyl chloride, then oxidizing them in periodic acid, and finally treating them first with sulphurous acid (at pH 3) and second with a solution of any basic fluorescent dye at a pH between 2 and 3. In sections treated in this way, nearly all periodate-oxidized mucosubstances emit a much more intense fluorescence than when treated with Kasten's Schiff-type reagents; and furthermore, that such mucosubstances fluoresce at all lends support to the theory that aldehydes react with Schiff's reagent proper not through N-sulphinic acids, as has been commonly supposed, but by reacting first with the sulphurous acid present in Schiff's reagent to form an intermediate alkyl sulphonic acid which then combines with basic pararosaniline (the essential ingredient of Schiff's reagent) to give the familiar magenta-coloured product.     

Preparation of a new red dye from Janus black and its use in the staining of DNA-aldehyde molecules

This communication presents a method for the preparation of a new red dye from an aqueous solution of Janus black by adding NHC1 and sodium thiosulphate to it. This new red dye when used on acid-hydrolysed tissue sections reveals the presence of red nuclei when sections after staining are dried between folds of filter paper, differentiated in n-butanol, cleared in xylene and mounted. Similarly stained sections when treated with SO2 water show partial leaching of the dye from the nuclei. Tissue sections when treated with cold concentrated phosphoric acid for 20 min and then stained with an aqueous solution of Janus black reveal the presence of orange-red nuclei. The new red dye obtained from Janus black does not respond to treatment under UV rays. The in vitro absorption data of the red dye indicate peaks at 210, 270 and 545 nm. The in situ absorption spectra of nuclei stained with the new red dye following Feulgen procedure reveal the peak of maximum absorption at 560 nm and those of nuclei treated with cold concentrated phosphoric acid and then stained with this red dye reveal peak at 530--540 nm. Some relevant points raised out of this investigation have been discussed.     

Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523).     

Modification of Cunningham's method for the detection of proteases in tissue sections.

A modification of Cunningham's method to show the protease activity in fresh tissue sections on gelatin film is proposed. The diazotization of the gelatin film in two successive baths of Fast blue B salt sharply increases the colour intensity of the film, improving the contrast between the intact zones and the enzymatic lysis zones of the substrate. The various washings of the stained film have been simplified. After incubation, the histological staining of the sections by solid nuclear red is an improvement over the much more tricky Feulgen reaction, combined with PAS, used by Cunningham.     

A technique for studying one and the same section of a cell in sequence with the light and electron microscope

Methods for mounting and staining relatively thin sections on electron microscope grids, in order that one and the same section of a cell can be photographed in sequence with the light and electron microscope are described. Toluidine blue is used as a stain and hexachlorabuta-1,3 diene as a medium which enables the grid carrying the stained sections to be temporarily mounted under a coverslip and examined with an oil-immersion lens. Results obtained with pollen mother cells of Fritillaria lanceolata at zygotene are illustrated.     

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