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Electron Microscopy Sciences

Technical Data Sheets

Bodian's Protargol Method

EMS Catalog #26693

Fixation

Formalin - Alcohol - Acetic Acid

Sections

Paraffin At 6 Microns

Staining

  1. Deparaffinize and hydrate to distilled water.
  2. 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.
  3. Wash by dipping in distilled water
  4. Reduce in hydroquinone solution for 5-10 minutes. Wash thoroughly in distilled water, three changes.
  5. Tone in Gold Chloride, 1.0% for 5-10 minutes. Rinse in distilled water, three changes.
  6. Develop in oxalic acid 2% until background is gray and nerve fibers appear clearly, 3-5 minutes. Rinse in distilled water, three changes.
  7. Place in sodium thiosulfate 5% 2-5 minutes. Rinse in distilled water.
  8. Counterstain with aniline blue solution 2 or 3 quick dips.
  9. 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.
  10. Dehydrate rapidly in absolute alcohol and clear in xylene, two changes each.
  11. Mount.

Staining Results

Nerve Fibers 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 color 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.

Online Ordering

Protargol Solution 1% Aqueous, Cooper Shot, Gold Chloride 1% Aqueous, Aniline Blue Solution, or Lissamine Fast Red Solution, Oxalic Acid 2% Aqueous, Sodium Thiosulfate 5% Aqueous, Phosphomolybdic Acid 1% Aqueous, Artrazine Solution 1.5% and Hydraquinone Reducing Solution for Bodian's Protargol Method are available online from the EMS Catalog. For ordering or product information, click here.