Methyl green-pyronin as a stain for autoradiographs of plant material.

Abstract

Methyl green-pyronin staining of autoradiographs successfully followed photographic processing. Stripping film was used, and staining was postponed until after film exposure to avoid extraction of labeled cellular constituents during the staining process. The transparency of the stain facilitates the localization of silver grains. Prior to application of the film, sections may be treated with nucleases. However, mild acid treatments, such as optimum hydrolysis for the Feulgen reaction, greatly reduce the intensity of the methyl green nuclear stain without removing a major DNA fraction. This result indicates that methyl green staining of DNA loses its specificity rather easily. With adequate controls, however, both stains can indicate the relative abundance of each nucleic acid. The staining procedure is: Autoradiographs are taken to water either from a dry state by means of a graded alcohol series or directly from the last photographic wash. Slides are transferred to 2 changes of dilute McIlvaine's buffer (1:10) at pH 4.2 for 5 min each, then stained 15 min in a solution consisting of 80 ml of the diluted buffer and 20 ml of a 2% aqueous solution of methyl green (Nat. Aniline), previously extracted 3-4 times with an equal volume of chloroform; dissolved in this solution is 0.5 gm of pyronin Y (G. T. Gurr) Slides are rinsed quickly in 2 changes of water, blotted and allowed to air dry. Typical results are: nuclei and chromosomes—blue; cytoplasm—pink; nucleoli—deep pink-red; and cell walls—pale blue.