Wilder Modification of the Bielschowsky Stain for Reticulum

EMS26392

Components:

Silver Nitrate 10.2% Aqueous

Ammonium Hydroxide, concentrated

Sodium Hydroxide 3.1%

Formalin 40%

Harris' Haematoxylin

Nuclear Fast Red

Phosphomolybdic Acid 10%

Uranium Nitrate 1%

Gold Chloride

Sodium Thiosulfate 5% Aqueous

Van Gieson's Solution

Fixation:

10% Buffered Neutral Formalin or Russell’s Zenker

Sections:

Cut paraffin at 6µm sections.

Staining Procedures:

1. Deparaffinise and hydrate slides to distilled water.
2. Oxidise in Phosphomolybdic Acid, 10% for 1 minute. Rinse well in distilled water or sunning water to prevent the cells from holding yellow colors.
3. Sensitise in Uranyl Nitrate, 1% for 1 minute or less. Rinse 10 – 20 seconds in distilled water.
4. Stain in fresh Ammonical Silver for one minute (changing solution frequently). The solution may be prepared as follows:
   1. Add concentrate Ammonium Hydroxide to 5.0ml of Silver Nitrate, 10.2% drop by drop, until the forming precipitate is almost dissolved.
   2. Add 5.0ml of Sodium Hydroxide, 3.1% and just dissolve the resultant precipitate by slowly adding concentrated Ammonium Hydroxide dropwise. Bring volume up to 50ml with distilled water.
   3. Use at once or filter and store in refrigerator in amber bottle.
5. Dip quickly in 95% alcohol and transfer to freshly prepared reducing solution. Prepare the reducing solution as follows:
   1. Mix 50.0 ml distilled water, 1.5ml Uranyl Nitrate, 1% and 0.5ml of  Formalin, 40% neutralised.
   2. Prepare just before use and change frequently during the process.
6. Rinse in distilled water.
7. Tone in Gold Chloride, 0.2% approx. 1 minute, until the yellow brown colour leaves the sections and they turn lavender. Check the slides at this point individually with a microscope to prevent over toning; this could lead to red sections.
8. Rinse in distilled water and transfer to Sodium Thiosulfate, 5% – approx. 1 minute – to fix the colour. Wash in tap water.
9. Counter-stain in Nuclear Fast Red if desired, rinsing well in distilled water. Alternatively  Harris' Haematoxylin with either Van Gieson's or phloxine can be used as a counterstain.
10. Dehydrate in 95% and 100% alcohol, 2 changes and clear in xylene, 2 changes.
11. Mount. If celloidin is used, clear in oil of origanium followed by 25% alcohol, usually mounted in balsam with Permount.

Stain Results:         

References:

Wilder, H.C., Amer. J. Clin. Path., 11:817, 1935.

Mallory, F.B. Pathological Technique, Phil., W.B. Saunders & Co, c. 1938

AFIP Manual of Histological Staining Techniques, NY, McGraw-Hill Publ., c. 1968/3rd ed. P.92