Studies on nucleic acid metachromasy. II. Metachromatic and orthochromatic staining by toluidine blue of nucleic acids in tissue sections.

Acrolein-fixed, polyester wax-embedded tissue sections showed excellent preservation of light microscopic architecture and, when stained with toluidine blue, intense color contrast between DNA, which stained orthochromatically, and RNA, which stained metachromatically. This method has practical value for differentiating DNA from RNA in the same section. The color contrast was impaired by substituting formaldehyde for acrolein or paraffin for polyester wax, and was negligible in tissues fixed in formaldehyde or Carnoy's fluid and embedded in paraffin. Quality of structural preservation paralleled degree of color contrast. Metachromatic staining can be analysed, by the quantitative parameters of Bradley and colleagues, to provide inferences regarding the conformation of biopolymers in tissue sections. Comparison of the nucleic acid color contrasts in toluidine blue-stained sections with titrations of fixative-treated nucleic acids against toluidine blue in solution indicated a greater difference in conformation between DNA- and RNA-protein in acrolein-polyester sections than between acrolein-treated free DNA and RNA in solution. This is supported by recent evidence that the conformation of ribosomal RNA is quite different in whole ribosomes from that assumed by the same RNA free in solution. The acrolein-polyester method may enhance color contrast by providing superior preservation of ordered nucleoprotein conformations.