Bodian's Protargol Method

Created by Kathleen Patrick, Modified on Fri, 8 Nov at 12:26 PM by Kathleen Patrick

EMS26693

Components

Protargol Solution, 1% Aqueous

Cooper Shot

Gold Chloride, 1% Aqueous

Aniline Blue Solution

Lissamine Fast Red Solution

Oxalic Acid, 2% Aqueous

Sodium Thiosulfate, 5% Aqueou

Phosphomolybdic Acid, 1% Aqueous

Tartrazine Solution, 1.5%

Hydraquinone Reducing Solution

Fixation

Formalin - Alcohol - Acetic Acid

Sections

Paraffin At 6µm

Staining

Deparaffinise and hydrate to distilled water.
Place slides in Protargol Solution and add 4-6g clean copper shot per 100ml of solution. Let stand at 37°C for 48 hours or 60°C for 4-16 hours. Staining is complete when sections are golden brown.
Wash by dipping in distilled water
Reduce in hydroquinone solution for 5-10 minutes. Wash thoroughly in distilled water, three changes.
Tone in Gold Chloride, 1.0% for 5-10 minutes. Rinse in distilled water, three changes.
Develop in oxalic acid 2% until background is gray and nerve fibers appear clearly, 3-5 minutes. Rinse in distilled water, three changes.
Place in sodium thiosulfate 5% 2-5 minutes. Rinse in distilled water.
Counterstain with aniline blue solution 2 or 3 quick dips.
Alternatively Counterstain with lissamine fast red for 5 minutes. Place in phosphomolybdic acid 1% for 5 minutes. Rinse briefly in distilled water. Place in tartrazine solution for 5 minutes. Rinse briefly in 95% alcohol.
Dehydrate rapidly in absolute alcohol and clear in xylene, two changes each.
Mount.

Staining Results

Nerve Fibres	Black
Myelinin, Muscle, Erythrocytes Lissamine Fast Red Counterstain	Red
Background-Lissamine fast red Counterstain	Yellow
Background-Aniline Blue Counterstain	Blue

Copper shot is good as long as it retains a metallic luster. If a dull oxide film forms discard the Copper Shot or clean by immersing in 30% Nitric Acid (30ml of conc. Nitric Acid added slowly and carefully to 70ml of distilled water) until the surface film or oxides is removed. The Copper will be bright in colour and all dark areas of oxide removed. Rinse quickly in D-H20, several changes, followed by one or two changes of alcohol or acetone. Dry in a low temperature (approx. 500°C) oven for 15-30 minutes. Store in a tightly sealed bottle.

References

Luna, L.G., (ed.), Manual of Histologic Staining Methods of the AFIP 3 rd edition, McGraw Hill, NY, c 1968, p. 195.

Clark, G., (ed.) Staining Procedures , 3 rd edition, Williams & Wilkins co., Baltimore c. 1973, 98.