All solutions of these dyes should be filtered before using, and should be kept covered to avoid evaporation and subsequent precipitation.
1. Formalin fixation 24 hours; cut on freezing-microtome.
2. Place sections in 70 per cent alcohol.
3. Stain in the simple solution 20-30 minutes; in the acetone or alkaline alcoholic solutions 2-3 minutes.
4. Wash in 50-70 per cent alcohol, differentiating as needed.
5. Transfer to water; thence to slide; blot, and mount in glycerin gelatin.
When a nuclear counterstain is desired, put the sections in water after 4; then stain in hæmatoxylin; differentiate quickly in acid alcohol; wash in water; place in weak ammonia or lithium-carbonate solution; wash in water; transfer to slide; blot; mount in glycerin gelatin.
Sudan III and scarlet R stain the smallest particles of fat yellowish-red to deep scarlet; scarlet R on the whole gives the best results. The contrast with the blue nuclei when stained with hæmatoxylin gives beautiful preparations.
3. Staining of Fat with Indophenol.
Stain sections with lithium-carmine; wash; then stain 20 minutes in a saturated solution of indophenol in 70 per cent alcohol. Fat blue; nuclei red.