4. Staining of Fatty Acids and Soaps.
a. Benda’s Method. Fix in 10 per cent formol. Transfer tissue to Weigert’s copper-fluorchrom mordant (neutral copper acetate 5 grms., fluorchrom 2.5 grms., water 100 cc.; boil and add 5 cc. of 36 per cent acetic acid) in the incubator for 2-4 days. Cut on the freezing-microtome. Stain sections in sudan III or scharlach R, and then in hæmatoxylin. Nuclei are blue, normal fat red, necrosed fat green due to formation of fatty acid copper salt. Soaps give the same reaction when converted into insoluble salts by fixing in formol saturated with calcium salicylate. Through comparison of tissue hardened in this way with another portion fixed in formol alone soaps and fatty acids may be differentiated.
b. Smith’s Method. Stain in concentrated water solution of Nile blue sulphate for 10 minutes. Fat stains red, nuclei dark blue, protoplasm light blue, fatty acids dark-blue. Differentiate in 1 per cent acetic acid; wash in water; mount in glycerin-gelatin.
X. FIBRIN. Fibrin stains with the acid aniline dyes, except in areas of necrosis containing diffused chromatin, under which conditions it stains deep blue with hæmatoxylin. In Van Gieson’s mixture it stains yellow or brownish; in Mallory’s reticulum stain it stains red, and with Mallory’s chloride of iron hæmatoxylin it is grayish to dark blue. The best selective method by far is Weigert’s, and it is the only really practical method giving a good differentiation.
1. Weigert’s Fibrin Stain.
I have obtained the best results by making this stain as follows: 10 cc. of aniline oil and 100 cc. of water are shaken together violently for several minutes, and then filtered through a moist filter. The filtrate must contain no drops of aniline. Add to the filtrate sufficient dry gentian-violet or methyl-violet to produce a metallic shimmer on the surface of the solution after the dye is dissolved by shaking. The solution will keep for several months.
Weigert advised the use of two stock solutions, I (absolute alcohol 33 cc., aniline oil 9 cc., methyl violet in excess) and II (saturated water solution of methyl violet). These solutions will keep for years. When ready to use stain take 3 cc. of Sol. I and 27 cc. of Sol. II. This staining mixture will keep for about 2 weeks.
1. Fix in alcohol, formol, acetone, mercuric chloride or Müller’s. Imbed in celloidin or paraffin; the latter preferably. Mount sections on cover-glass with albumin fixative. Celloidin sections must be fastened to slide by thin film of celloidin to prevent shrinkage. Sections fixed in chromic mixtures (and sometimes after formol fixation) must be oxidized in potassium permanganate and then reduced in oxalic acid to give good results. (Transfer sections to a 1 per cent solution of potassium permanganate to which 2 volumes of water have been added; oxidize for 10 minutes; then wash in water, and reduce for several hours in a 5 per cent water oxalic acid solution.)
2. Wash in water.
3. Stain in lithium carmine; differentiate in acid alcohol; wash thoroughly in water.