Weigert's Staining Methods

WEIGERT'S STAINING METHODS are used in the processing of the nervous system (for isolated staining of myelin, neuroglia), elastic fibers, fibrin, bacteria, and for nuclei. 1. For myelin staining, prolonged (at least a week) hardening of pieces of the nervous system in Müller's fluid or in the so-called Weigert's mordant for 4–5 days is necessary (Kali bichromici 5 parts, Alumen chromicum 2 parts, water 100 parts). The duration of stay in fixing fluids depends on the size of the pieces. Processing with alcohols in ascending concentration in the dark, embedding in celloidin (paraffin is also possible). Sections are placed for 24 hours in 1% Cuprum aceticum, then, without rinsing, transferred for 24 hours at 37°C into Weigert's hematoxylin (hematoxylin 1 part, absolute alcohol 10 parts, water 100 parts); they are differentiated after washing in the following Weigert's solution: borax 4 parts, potassium ferricyanide 5 parts, water 200 parts; myelin sheaths stain black or dark blue, erythrocytes and fibrin fibers stain black; after this, the sections are thoroughly washed in plain water, dehydrated, and mounted in balsam. The Weigert-Pal method is also very commonly used—a modification of the previous one, the difference being only in the differentiation; after hematoxylin, sections are transferred to a 1/4% solution of Kali hypermanganici (for 30 sec.–1 min.), then, after rinsing, into a solution of Ac. oxalici 1:100 + Kali sulfurosi 1:100 (both solutions are mixed before use); after differentiation, a more relief-like staining is obtained, but with different shades. For neuroglia staining, preliminary fixation of very small pieces for 8 days in the following solution is necessary: Alumen chromicum 2.5 parts, Ac. aceticum 5 parts, Cuprum aceticum 5 parts, water 100 parts; then embed in paraffin or cut on a freezing microtome; then the following processing: 10 min. in 1/4% Kali hypermanganici, rinse; for 2–4 hours in a solution: 5% Chromogen 5 parts, Ac. formic. 5 parts, water 100 parts; for 2–8 days in an aqueous 5% solution of Chromogen, staining in a saturated alcoholic solution: Methyl-violett 100 parts + 5% Ac. oxalicum 5 parts, washing in 0.9% NaCl, processing for several minutes with a saturated solution of iodine in Kali iodatum, differentiation in xylene + aniline (oil), mounting in balsam. With successful results, elective staining of the neuroglia in a bluish-lilac color is obtained; the method is very difficult and unreliable, as simultaneous staining of connective tissue is possible. 2. Elastic tissue according to Weigert's method is stained with resorcin-fuchsin, otherwise known as fuxelin: to 200 cc of 1% aqueous basic fuchsin, add 4 g of resorcinol and heat in a porcelain dish, add 25 cc of Liquor ferri sesquichlor. (= 29% FeCl₃) and boil for another 2–5 min. Allow to cool, filter off the resulting precipitate and place it together with the paper filter into the same porcelain dish, pour in 200 cc of 95° alcohol, heat to boiling while stirring, allow to cool and filter, add 4 cc of hydrochloric acid (sp. gr. 1.121 = 25% HCl). The solution stains well for only 4–5 weeks. Sections are stained for 10–20 min., then differentiated with 95° alcohol and mounted in Canada balsam. Elastic tissue fibers are dark blue, almost black, the background is colorless, nuclei can be pre-stained with carmine. 3. Hematoxylin is a good nuclear stain. Two solutions are prepared: I. - hematoxylin 1 g in 100 cc of 96% alcohol; II. - 4 cc of Liquor ferri sesquichlorati + 1 cc of hydrochloric acid (official) + 95 cc of water. Immediately before use, mix equal parts of solutions I and II. If possible, transfer thin sections from water into the stain for 1–5 min. Then wash with tap water and mount in balsam. Additionally, one can stain according to Van Gieson.