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THE EFFECT OF METHYLATION ON BASOPHILIA
1 National Institutes of Health, Bethesda, Md.
A mixture of 0.05 N HCl and methanol is capable of completely inhibiting thionin and azure A metachromasia of intestinal mucin, cartilage and mast cell granules after treatment for 2 hours at 58°C. or 48 hours at room temperature. Slight reduction is noted after 2 weeks at 3°C.
Cytoplasmic basophilia is completely destroyed after 4 hours of methylation at 58°C. or 48 hours at room temperature. Pancreatic acinar cell cytoplasm appears more resistant, requiring 8 hours at 58°C. and longer than 9 days at room temperature. Methylation at 3°C. is ineffective in inhibiting this basophilia even after 2 weeks.
Nuclear and nucleolar basophilia with aniline dyes (azure A and thionin) requires 24 hours of methylation at 58°C. or 14 days at room temperature before there is complete destruction. There is no effect produced by methylation at 3°C. for 2 weeks. Alum hematoxylin staining of these structures is unaffected by the methylation procedure, indicating the different nature of action of this dye complex from the aniline dyes used.
Reversal of the methylation procedure or demethylation can be accomplished with 0.5% KMnO4 for 
hour at room temperature provided the methylation has not been too prolonged. Other oxidants such as periodic, peracetic and chromic acids as well as aqueous HCl and alkali (borax solution) fail to reverse the blockade.
The substitution of 0.1 N HCl for the usual 0.05 N HCl in the methylation procedure brings about effective blockade in approximately the time required by the 0.05 H NCl methanol.
Despite the removal of cytoplasmic and nuclear basophilia by this method sections treated concomitantly and stained with the Feulgen nucleal procedure demonstrate strong Feulgen reactions indicating selectivity for the acid portion of the nucleic acid involved.
Methylation after fixation of tissue in a chromate containing fixative (Zenker acetic) requires more prolonged treatment to inhibit cytoplasmic basophilia than is required after Formalin or 95% alcohol fixation. Otherwise the reactions occur similarly after either of the three fixatives.
The positive periodic acid-Schiff reaction of colonic mucin, mixed tumor stroma and cartilage is completely inhibited by prior methylation for 96 hours at 58°C. and the reaction of gastric and small intestine mucins is only moderately reduced at this period. At room temperature no alteration is observed after methylation for 21 days. The positive reaction of human mast cell granules is abolished after 72 hours of treatment at 58°C. and is only moderately affected after treatment for 21 days at room temperature.
Submitted on September 10, 1953
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