Formol, mercuric chloride and Zenker’s all give good results. Paraffin imbedding, with staining of sections on the cover-glass (albumin-fixative method), is the best method of staining for permanent mounts, although good preparations can be obtained by the use of the freezing-microtome. Carbol-xylol cannot be used for clearing. Nuclei are blue, protoplasm pale blue, amyloid ruby-red.
As a specific reaction for amyloid and mucin this method has been used in my laboratory for the last ten years in preference to any other. The stains are permanent if not exposed to the action of light.
Thionin, toluidin-blue, polychrome-methylene-blue, and other metachromatic dyes are also used to give similar reactions with amyloid, but are not as satisfactory as the kresyl-echt-violett method. Amyloid may also be stained with scarlet R or sudan III, according to the method of Herxheimer, but the results are rarely satisfactory.
II. ATROPHY. Good pictures of atrophic tissues are obtained with formol-Müller’s, mercuric chloride or Zenker’s fixation, and staining with Van Gieson’s, to bring out the stroma which is usually relatively or absolutely increased. In the case of pigment-atrophy the sections should be very thin and stained with alum- or lithium-carmine.
III. CALCIFICATION. Deposits of lime-salts appear in fresh tissue as gritty, refractive areas that are bright and shining by reflected light, and dark by transmitted. They are soluble in acids, solution of the carbonate being accompanied by the formation of bubbles of carbonic acid gas. Hæmalum and the alum-hæmatoxylins show a specific reaction with the phosphates and carbonates of lime, giving them a deep blue or reddish-violet stain. Fresh calcification usually stains diffusely blue; older deposits are deep-blue about the borders of the deposits, lighter or unstained in the center of the mass. Tissues containing much calcium must be decalcified before imbedding. If the process of decalcification is not carried too far the specific staining reaction is not lost.
v. Kossa’s Silver Method for Calcium Phosphate.
1. Fix in alcohol or formol; imbed; cut.
2. Place section in 1-5 per cent silver nitrate solution, and expose to daylight 5 minutes to 1 hour.
3. Wash in distilled water.
4. Transfer section to a 5 per cent solution of sodium hyposulphite, to remove excess of silver nitrate.