3. Wash thoroughly in water.
4. Differentiate in 95 per cent alcohol until connective-tissue is colorless.
5. Dehydrate in absolute alcohol; xylol; balsam.
Keratohyalin red, keratin dark violet; nuclei blue-violet, plasma light blue-violet.
IX. FAT. When alcohol has been used in the preparation of the tissue, the fat-contents of the latter are dissolved out, and their presence can alone be told by the presence of vacuoles. When osmic acid is used as a fixing agent the oleates and oleic acid are blackened. The tissue should then be washed in running water and cut upon the freezing-microtome, or it may be imbedded in celloidin or paraffin if this is done as quickly as possible to prevent the loss of the fat. Chloroform or benzene should be used in place of xylol, as the last-named dissolves out the fat. Safranin should be used as a stain after fixation with any fluid containing osmic acid. Frozen sections are to be mounted in glycerin-gelatin; when balsam is used it should be warm melted Canada balsam without xylol. Formol fixation preserves fat, and tissues so fixed may be cut on the freezing-microtome and the sections stained with osmic acid, sudan III or scharlach R, with nuclear counterstaining when desired. For the demonstration of fat-embolism, fatty degeneration or fatty infiltration the following methods are advised:—
1. Staining of Fat with Osmic Acid.
1. Fix in formol for 24 hours.
2. Wash; freeze; cut.
3. Place sections in 1 per cent osmic acid, Flemming’s or Marchi’s fluid for 1-24 hours.
4. Wash in water, changing frequently.