Procedure: Oxidize sections in 0.4% periodic acid for 30 minutes at 57°C. Wash in running tap water or sodium sulfite solution (to avoid a general pink background). Rinse in three times in distilled water. Schiff's Reagent for 15 minutes. Wash well in running tap water. Rinse in distilled water. Counterstain with Gill's Hematoxylin for 10 minutes. Wash well in running tap water. Dehydrate using 96% and 100% ethanol. Clear in xylene and coverslip. Results:

Nuclei - blue Glycogen - violet / red Basement membranes - violet / red Mucine - violet / red

Solutions: Gill's Hemotoxylin

• Hemotoxylin (C.I. 75290) - 6 gm
• Sodium Iodate - 0.6 gm
• Aluminum Sulfate - 52.8 gm
• Distilled Water - 690 ml
• Ethylene Glycol - 250 ml
• Glacial Acetic Acid - 60 ml

Schiff's Reagent

Mix 0.5 gm of Pararosaniline (C.I.#42500) into 15 ml of 1N Hydrochloric Acid. Mix 0.5 gm of Potassium metabisulfite into 85 ml of Distilled water. Add the second solution to the first and store for 24 hours in a dark place. Bleach the resulting solution with 200 mg of bone charcoal and filter. Store the colorless solution at 4°C.

Sodium Sulfite Solution

• 0.5 gm of sodium sulfite.
• 100 ml of distilled water.

Citation:

Gerrits, P. O. and Smid, L., "Staining Procedures for Tissues Embedded in 2-Hydroxyethyl Methacrylate", Heraeus Kulzer