Bielschowsky's Method

Bielschowsky's method, for staining neurofibrils. 1. Fixation in 10–20% formalin for 3 days or more. The optimum is from 3 to 9 weeks. For shorter periods, it is better to fix at a temperature of 37°. 2. Washing for 2–3 hours in running water and 24–48 hours in distilled water. 3. Cutting on a freezing microtome at 5–10 µ. Sections are transferred to distilled water. 4. Using a glass rod, sections are transferred for 24 hours into 2% chemically pure silver nitrate. Usually, a 10% solution of silver nitrate is prepared, which is then diluted with distilled water. 5. Processing through ammoniacal silver and reduction with formalin. Glassware—measuring cylinders and crystallizing dishes (washed with technical sulfuric acid). Ammoniacal silver is prepared ex tempore. To 20 cc of 2% AgNO₃, add 5 drops of 40% NaOH; a brown precipitate forms, which is dissolved by adding ammonia drop by drop; NH₄OH (Liquor ammonii caustici triplex), just like the NaOH solution, must be changed frequently. The art of preparation lies in not adding a single extra drop of ammonia. For this, it is better to immediately add the amount of ammonia that experience has shown is necessary for dissolution. During dissolution, one should assist the process by lightly shaking the cylinder. According to the previous formula, 5 cc of 10% AgNO₃ was taken and 5 (or 4) drops of 40% sodium hydroxide were added. Since the precipitate dissolves better in an excess of ammonia, one can immediately add 5 large drops of ammonia, not shake the liquid, and, leaving the light ring of ammonia that forms at the top intact, lift the clouds of precipitate into this ring with light shaking. After the precipitate dissolves, distilled water must be added up to 40 cc (according to the previous formula—from 5 cc to 20 cc). Reduction procedure: take 4 crystallizing dishes: 1) with distilled water, 2) with ammoniacal silver, 3) with distilled water, and 4) with 20% formalin. Sections are transferred one by one for a few seconds into distilled water, then for about 5 minutes into ammoniacal silver, where they turn even browner and flatten out, then for a few seconds again into distilled water, and finally into formalin. One can transfer them after the ammoniacal silver not through one, but through several dishes with distilled water, but it must be done quickly. In formalin, the sections darken; reduction proceeds very rapidly.

6. Gold toning and fixing (not mandatory, but desirable): 1) distilled water; 2) gold chloride, to 10 cc of distilled water add 3–5 drops of 1% gold chloride (yellow tone), keep the section until a steel color appears; 3) 5% sodium thiosulfate for 20–60 seconds; 4) water. 7. Alcohols of increasing concentration, carbol-xylene; xylene and Canada balsam. Modifications of Bielschowsky's method: 1. To obtain special cylinders, sections can be placed for 24 hours in pure pyridine, then washed for several hours in distilled water, then 2% silver nitrate, etc. 2. For impregnation in blocks: 1) after formalin fixation and washing, the block is placed for 3 days in pure pyridine at room temperature; 2) distilled water for several hours; 3) 3% silver nitrate at 36° for 3 days; 4) quickly—distilled water and then ammoniacal silver for 24 hours; dilution with ammoniacal silver is done up to 60 cc; 5) distilled water for up to 1 hour; 6) 20% formalin; 7) water; embedding in paraffin; 8) cutting; removal of paraffin; 9) gold toning, fixing, etc. Instead of formalin, Bielschowsky also recommends for reduction 20% grape sugar or the following mixture: 75.0 cc of grape sugar (30%); 75 cc of Rochelle salt (10%); 20 cc of potash (10%) and 5 cc of pure formalin. For all procedures, it is desirable to use bidistilled water.