Staining of acid-hydrolysed tissue sections with Schiff-type dye-reagents under UV rays

This paper reports on the use of some Schiff-type dye-reagents prepared with oxalic acid and sodium thiosulphate. Some of the dyes, such as azure A, azure B, toluidine blue O, thionine, brilliant cresyl blue, and methylene violet were also prepared with oxalic acid and sodium thiosulphate but fortified with disodium hydrogen phosphate. It has been found that all these dye-reagents, barring neutral red-SO2, when used on acid-hydrolysed mammalian tissue sections under UV rays yield far better staining of the DNA-aldehyde molecules than is possible in controls stained under normal laboratory conditions. Sections stained with any of the dye-reagents under UV rays do withstand treatment in SO2 water or acid water without showing any leaching of the dye from the nuclei. Aqueous solutions of all these dyes can also be used to stain DNA-aldehyde molecules of acid-hydrolysed tissue sections. But sections stained with aqueous solutions of the azures, safranine, phenosafranine, and methylene violet when treated with SO2 water reveal considerable leaching of the dye from the nuclei. However, leaching does not occur when these dyes are used as SO2-containing dye-reagents under UV rays. It has, therefore, been concluded that in the case of staining with the azures, safranine, phenosafranine, and methylene violet, containing SO2, the reaction under UV rays is of Feulgen-type. In the case of staining with toluidine blue-SO2, thionine-SO2, and brilliant cresyl blue-SO2 under UV rays, the mechanism of staining is also of Feulgen-type but it is due to liberation of the dye molecules from their more or less colourless states that react with the DNA-aldehyde molecules producing nuclear colouration as though these dyes are in aqueous solution. This interpretation is based on the fact that sections from which RNA has been extracted selectively with cold concentrated phosphoric acid when treated with these dye-reagents under UV rays also stain DNA-phosphate groups. The increased staining intensity of the nuclei produced with the majority of the dye-reagents under UV rays as compared with control sections stained under usual laboratory conditions has been considered to be due to electronic excitation of the dye molecules thus facilitating binding between more molecules of DNA-aldehyde and of dye. Possible significance of these findings has been discussed.