The effect of histochemical methylation on the phosphate groups of nucleic acids: interpretation of the absence of nuclear basophilia.

The effect of hot methylation (hydrochloric acid-methanol)) on nuclear stainability was investigated in order to determine whether the progressive loss of basophilia is due to methylation of the diester phosphate groups of nucleic acid. DNA spots on filter paper were unchanged in their stainability towards Toluidine Blue even after methylation for 4 days, while RNA, chondroitin sulphate and hyaluronic acid lost their affinity for this dye after 4 h methylation. In formalin-fixed sections, methylation for 4 h led to a loss of nuclear basophilia. There was no concomitant increase in nuclear relative to cytoplasmic stainability with Fast Green FCF at pH 9, as judged from the use of a comparison eyepiece, evaluation of colour transparencies or by microspectrophotometry. In contrast, extraction with trichloroacetic acid prior to or after methylation led to a much improved Fast Green staining of nuclei, comparable to the staining obtainable after treatment with trichloroacetic acid alone. In conclusion, there is no evidence that hot hydrochloric acid-methanol, as used in histochemistry, methylates the diester phosphate groups of nucleic acids. The loss of nuclear basophilia can be explained as a result of the excess positive charge on the chromatin following methylation of all the protein carboxyl groups. This effect is maximal after 3-4 h treatment with acid methanol at 60 degrees C. Further methylation leads to depolymerization and extraction of DNA. RNA is depolymerized in less than 4 h.