Snesarev Methods

Snesarev Methods. 1. Method for non-neuroglia: 1) fixation of brain tissue in formalin; 2) washing in running water and freezing of sections; the latter to be taken into distilled water; 3) each section is held for 30 seconds in a solution: 1% phosphomolybdic acid and an equal amount of distilled water; add 5-10 drops of a 1:1,000 solution of potassium carbonate; 4) rinsing in distilled water and mounting in a wide Petri dish on a slide coated with egg white-glycerin. Allow excess water to drain, dry around the section with a cloth, and blot with filter paper moistened in methyl alcohol+chloroform (1:2, 1:3); pour the same mixture over the section until it is strengthened and cleared; 5) pour May-Grünwald stain on the moist section (do not stain for long); 6) add 1-3 drops of distilled water and continue staining; 7) differentiation using diluted glacial acetic acid (1:2 parts water); 8) wash in water and restain with eosin for 3-5 minutes; 1 g of eosin in 150 g of distilled water and 2-3 drops of glacial acetic acid; 9) distilled water; 10) drying with filter paper; 11) quickly (1-2-3 seconds) dip into acetone; 12) xylene; 13) balsam. Gliofibrils are stained blue, cell nuclei in blue and pink, myelin in pink. Negri bodies are blue. The section can first be stained with eosin, and then the initial part of the procedure can be done. Modification of the Pappenheim method for frozen sections, especially for brain: 1) formalin fixation, washing, freezing of sections; the latter are heated in 33% acetic acid until vapors appear; 2) they are taken from distilled water and mounted on a slide coated with egg white-glycerin (by blotting with filter paper moistened in methyl alcohol+chloroform); 3) May-Grünwald stain (for about one minute); 4) add 1-2 drops of distilled water; 5) restain according to Giemsa (1 drop per 1 cm³); 6) quick wash and drying with filter paper; 7) into acetone for 1-2 seconds; 8) xylene; 9) balsam. The author recommends this method as a general one for different tissues. In the brain, Nissl granulation and Negri bodies are stained pink. Modification of the Jahnel method for spirochetes: 1) formalin fixation; 2) running water; 3) anhydrous pyridine for 1-2-3 days; 4) washing of pieces in running water, then in distilled water; 5) embedding in 24% gelatin (in 1% carbolic water) for 2 hours or more; 6) cool the gelatin, cut out pieces, fix them in formalin (for up to 6 days); 7) running water, freezing of sections; 8) distilled water and 96% alcohol for an hour; distilled water; 9) 5% uranyl nitrate at 37° in an incubator for 2-3 hours; 10) 2-3 portions of distilled water; 11) 1% silver nitrate for 3-6-12 hours; 12) reduction with 5% hydroquinone (to 5 cm³ add 15 cm³ of 70% gum arabic). Sections are taken into a Koch dish with 5 cm³ of 74% silver nitrate; 15 cm³ of 70% gum arabic is also poured in and the liquids are mixed by shaking, then the reducing liquid is added. (V. K. Beletsky.) 2) Method for argyrophilic fibers: 1) fixation in formalin (10-14-20%); 2) washing in running water; 3) several times in distilled water and embedding first in 12%, then in 24% solution of gelatin in 1% carbolic water, all the time in an incubator (from several hours to 2 days); 4) cooling of the gelatin and fixation in 5-10% formalin; 5) freezing of sections; 6) 4% solution of Alumen ferricum (iron alum) for two days without an incubator; 7) two to three portions of distilled water (10-15 minutes); 8) 10% silver nitrate - for a day. Then the sections are processed according to the Belyapovsky method, i.e., quick wash in distilled water and ammoniacal silver (3-5-10 minutes), quick wash in distilled water and reduction in 20% formalin; then distilled water, chloroauric acid, 5% sodium thiosulfate, distilled water and mounting in Canadian balsam. Preparation of ammoniacal silver: to 4 parts of 10% silver nitrate in a test tube add 3 drops of 40% sodium hydroxide (chemically pure), do not shake the precipitate, but pour on top 5 drops of ammonia (L. ammonii caust. triplex). Carefully shake the test tube so that the clouds of turbidity and the denser precipitate that rise in the liquid dissolve in the upper light ring of ammonia. If 5 drops are insufficient, add 1-2-3 more until dissolved. Then add distilled water to 20 parts. For dehydration, the author recommends first mounting the sections on a slide with egg white and then passing them through acetone, xylene and mounting in Canadian balsam. Modification of the same method for paraffin sections: 1) deparaffinization of sections, alcohols, water; 2) thorough washing with distilled water; 3) slides with sections are passed through 12.5% and 24% gelatin (in 1% carbolic water) in an incubator; 4) allow the gelatin to harden in an even layer on the side of the section; 5) fixation - formalin 5-10%; 6) rinse with distilled water and place slides with sections for 2 days in 4% Alumen ferricum. Then follows - distilled water, for a day 10% silver nitrate and the rest according to Belyapovsky.