  |
MICROWAVE RHODANINE COPPER METHOD
Click the Section Headings (Blue) to Expand/Collapse Material
|
|
|
|
|
|
|
Red-brown cytoplasmic granules are copper deposits in the this liver from a patient with Wilsons diasease [top left]. Note the bile (plug) remains green (left panel). Courtesy of Dr. Zhenhong Qu) |
|
Positive stain for copper is strongly indicative of Wilsons disease, although negative stain does not exclude this diagnosis.
See also ____ and _____ stains for copper.
FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 5 μm.
SOLUTIONS:
0.4% Rhodanine Solution (prepare fresh)
5 – (p-dimethylaminobenzylidene) rhodanine ------------------------------------- 0.024 gm
Absolute alcohol ----------------------------------------------------------------- 6.000 ml
1% Sodium Acetate Solution
*Sodium acetate trihydrate ------------------------------------------------------ 1.0 gm
Distilled water ------------------------------------------------------------------ 100.0 ml
*If anhydrous sodium acetate is used, use 0.6 gm.
Rhodanine Solution (Working)
Rhodanine solution (stock) ------------------------------------------------------ 5.0 ml
1% sodium acetate -------------------------------------------------------------- 45.0 ml
Filter the rhodanine solution through Whatman #4 filter paper (70 mm in diameter) into the 1% sodium acetate.
0.4% Sodium Borate
Sodium borate, (Na2B4O7 ∙ 10 H2O) ------------------------------------------ ----- 0.4 gm
Distilled water --------------------------------------------------------------------- 100.0 ml
2. Place slides in the working rhodanine solution in a plastic Coplin jar, with the cap loosely applied, and microwave at power level 6 (360W) for 30 seconds. Agitate for about 15 seconds. Return the Coplin jar to the microwave oven and microwave at power level 6 for 35 seconds. Agitate for about 15 seconds and allow the slides to remain in this hot solution (about 80o C) for 5 minutes.
3. Return the Coplin jar to the microwave oven and microwave at power level 6 for 10 seconds. Agitate for about 15 seconds and allow the slides to remain in the hot solution for 5 minutes.
Copper -------------------------------------------------------- bright red or rust-red intracytoplasmic granules
Nuclei --------------------------------------------------------- blue
We have found this stain to be both sensitive and specific and to allow for a semiquantitative evaluation of the amount of copper present (see Irons, et al). This microwave method may not always give as good results as the overnight method which is done in a 37o C oven. A good positive control of this method is any fetal liver of the third trimester fixed in buffered neutral formalin for not longer than 24 hours prior to paraffin processing. Fetal liver of the third trimester contains copper within hepatocytes. Care must be taken not to over counterstain with hematoxylin which will mask the positive staining of the copper.
Wenger, J.B. and Luna, L.G.: A new method for compounding the rhodanine copper stain. Histo-Logic 18:50-51, 1988.
Brinn, N.T. and Goodfellow, B.C.: Metallic histologic impregnation using the microwave oven. Presented at the National Society for Histotechnology Symposium on October 21, 1985 in Crystal City, Virginia.
Irons, R.D., Schenk, E.A., and Lee, C.K.: Cytologic methods for copper. Arch. Path. Lab. Med. 101:298-301, 1977.
Lindquist, R.R.: Cytochemical method for the localization of copper. Arch. Path. 87:370-379, 1969.
Image Examples:
Diagnostic Application: