The following method obtained from "Diagnostic Histochemistry",
Zugibe, 1970 (C.V. Mosby Company)

Timm's Copper Stain-Slightly Modified

Fixation: Formalin fixed or fresh tissue.

Technique: Contiguous paraffin secrtions or cryostat sections may be
used.

Reagents:    Solution A
                      5.0% Silver Nitrate aqueous solution

                      Solution B
                      Hydroquinone----------2.0 g
                      Citric Acid---------------5.0g
                      Distilled H2O-----------100ml
                      This solution can be stored in the dark at 4 C
                       for one month.

Procedure:
              1. deparaffinize in two changes of xylene and hydrate
sections to water through graded alcohols.

               2. Place in 0.5% ammonium sulfide solution for five
minutes.

                3. Wash well in dH2O.

                 4. Treat sections with 0.1 N HCl for 2-3 minutes to
remove iron and Zinc sulfides (15% Trichloracetic acid may also be
used).

                  5. Rinse in dH2O.

                   6. Develop sections in freshly filtered solution
of 1 part A and 5 parts B. This converts copper sulfide to silver
sulfide. (I found approx. 3 mins. was sufficient for me).

                    7. Rinse in dH2O.

                     8. dehydrate, clear nad mount.

Results:
                Copper deposits are stained black.

Comments:
The following control procedures should be employed with contiguous
sections. (1) Omit step 4 to stain all heavy metals. (2) Omit step 6.
(3) Treat with 0.5% potassium cyanide before developing to remove
copper sulfide. (4) stain with Perl's method for iron.

Reference: Timm - Histochemie 2:332 - 341, 1961.
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Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
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Charlottetown, P.E.I.
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