Phosphoric acid-its use in the extraction of RNA: staining of DNA in mammalian tissue sections.
This communication presents highly satisfactory methods for the demonstration of DNA in animal materials. The method involves selective extraction of RNA with concentrated, 90% or 75% phosphoric acid of 5 degrees C for 20, 40 and 120 min, respectively, followed by staining with aqueous solutions of basic dyes, such as setoglaucine, setocyanine, pinakryptol green 2) and alcoholic aniline blue without SO2. Perfect blue nuclei were seen when staining was performed with aqueous solutions of setoglaucine and setocyanine at pHs 3.5 and 4.0 to 4.5, respectively. Sections of tissues from which RNA has been extracted and then hydrolysed in 6 N HCl at 28 degrees C for 15 min followed by staining with these dyes also revealed perfect colouration of the nuclei. Acid-hydrolysed sections when stained with alcoholic aniline blue-SW2 prepared with N HCl and sodium thiosulphate revealed nuclei of magenta colour, and sections from whcih RNA has been extracted and then hydrolysed in hydrochloric acid and stained with this dye-reagent revealed nuclei of purplish colour. Sections of tissues fixed in Carnoy, 10% buffered neutral formalin as well as paraformaldehyde were found to be most suitable for staining with these dyes. The in situ absorption spectra of nuclei stained with aqueous solutions of setoglaucine, setocyanine and alcoholic aniline blue without SO2, after extraction of RNA as well as those of nuclei in tissue sections from which RNA has been extracted and then acid-hydrolysed and stained with alcoholic aniline blue-SO2 have been presented. Also presented herein are absorption data of nuclei in tissue sections which wee hydrolysed in hydrochloric acid and then stained with alcoholic aniline blue-SO2. Some implications of these findings have been discussed.
D000814:Aniline Compounds; D000818:Animals; D002467:Cell Nucleus / Q000032:analysis; D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes; D010619:Phenazines; D010756:Phosphoric Acids; D012313:RNA / Q000302:isolation & purification; D051381:Rats; D012394:Rosaniline Dyes; D013194:Staining and Labeling / Q000379:methods*
D000814:Aniline Compounds; D004396:Coloring Agents; D005456:Fluorescent Dyes; D010619:Phenazines; D010756:Phosphoric Acids; D012394:Rosaniline Dyes; C033102:pinakryptol green; C033100:setoglaucine; C033101:setocyanine; D012313:RNA; C017006:aniline blue; D004247:DNA; C030242:phosphoric acid