Timm's Copper Stain

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From:Greg Dobbin <dobbin@Upei.CA> (by way of histonet)
To:histonet <histonet@magicnet.net>
Content-Type:text/plain; charset="us-ascii"

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

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.

              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

                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

                  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.

                Copper deposits are stained black.

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.
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 Unviversity Ave.
Charlottetown, P.E.I.
C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851

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