III. COMBINED NUCLEAR AND DIFFUSE STAINS.
The diffuse or plasma stains may be combined with the nuclear in one staining solution, or used in succession. The latter method gives better results. The nuclei are stained first, the diffuse stain being used after the washing-out following the use of the nuclear stain, except in the methylene-blue and eosin method in which to obtain the best results it is necessary to stain with the eosin first. Nuclear hæmatoxylin may also be followed by a combination of plasma stains, as in the Van Gieson’s mixture of picric acid and acid fuchsin, Delépine’s mixture of rubin and orange, White’s erythrosin and picric acid mixture, etc. In these mixtures the different affinities of the plasma-stains give rise to differential or selective staining effects.
a. Haematoxylin and Eosin.
1. Stain in any one of the hæmatoxylins.
2. Wash thoroughly.
3. Stain in dilute water or alcoholic eosin until section is bright rose-red.
4. Differentiate eosin-staining, as desired, by rapid or slow washing in water.
5. Dehydrate quickly in 80 and 95 per cent alcohols.
6. Clear quickly in carbol-xylol. (If carbol-xylol takes out eosin too rapidly add some of the dry eosin stain to it. Hæmatoxylin-stained sections can be placed in such eosin-carbol-xylol and will take up the eosin beautifully.)
7. Mount in balsam.