Methyl Green-Pyronin Staining: Effects of Fixation; Use in Routine Pathology

After formalin fixation and paraffin embedding, plasma cells and other py-roninophilic cells may be demonstrated as follows: The staining solution contains 4 ml of a 2% aqueous solution of methyl green, 6 ml of a 2% aqueous solution of pyronin Y, 2 ml of absolute ethanol, and Walpole's HCl-sodium acetate buffer at pH 3.8 to make a final volume of 100 ml. Staining is for 15–30 sec, differentiation by dipping twice, 1–2 sec each, in 2 changes of distilled water, next by keeping the slides in 2 changes of 96% ethanol for 30 sec each and finally by dipping them 6 times in each of 2 changes of absolute ethanol before clearing in xylene and covering in Canada balsam. Both the methyl green (Merck, C.I. No. 42585) and pyronin Y (National Aniline, C.I. No. 45005, Cert. No. NPy-20) stock solutions should be well extracted with chloroform. Sections of freeze-substituted tissues, frozen sections and smears are stained for 5 min or more in a similar solution made up with 0.1 M sodium acetate-acetic acid at pH 4.8, next the slides are dipped twice in 2 changes of distilled water and 6 times in each of 2 changes of 96% ethanol and 2 changes of absolute ethanol before clearing and mounting. The use of staining solutions at pH 3.8 and pH 4.8 provides for control of pyroninophilia.