Technique for the Complete Preservation of Supravital Stain of Neutral Red in Paraffin Sections

Abstract

The technique for the preservation of the supravital stain of neutral red in paraffin sections has been described by Mcjunkin 1 and Forkner. 2 Their methods enable one to observe most of the neutral red present in the cells; but a certain amount of the stain is inevitably lost in the process of preparation, and they require rapid dehydration and strict limitation of the size of the tissue blocks. The method described below has the advantage over those previously described, in that the entire neutral red stain within the cells is faithfully preserved, that the size of the blocks can be reasonably large, and that the different steps of staining can be carried out in a more leisurely and comfortable manner.

In this technique, advantage is taken of the fact that neutral red is only slightly soluble in aqueous or alcoholic solution containing mercuric bichloride. After the minimal amount of the bichloride required in each solution for the maximal insolubility of the neutral red is determined, any solution which will check further dissolution of neutral red from the tissue can be easily prepared from it.

The tissue to be studied is best stained by the direct injection method described by Forkner with only a slight modification through replacement of his Zenker-formalin by solution A (see below).

For carrying out this technique we use the following 4 different special solutions:

Solution A Fixing solution, prepared as follows:

Dissolve a small quantity of neutral red in 15 cc. of 40% formalin in a large flat dish. Add to it 85 cc. of Zenker's fluid. Most of the neutral red previously dissolved in formalin is now precipitated out. After a few minutes, during which time the mixture may be stirred with a glass rod, the mixture is filtered for use.