- Catalog order # K7284 - 4oz kit - $70.00
- K7288 - 8oz kit - $107.00
Silver Stain Kit Procedure (GMS)
APPLICATION
Mammalian Tissue
DESIGNED TO SHOW
Black oxidized Silver Deposits on opportunistic organisms and specific tissue elements
PURPOSE
For the detection of Fungus, Pneumocystis, Mucin, and Basement Membrane
SPECIMEN REQUIREMENTS
Fixation: Tissue: Any well fixed tissue, however, it is suggested to use formalin fixed tissue.
Sections: Cut Paraffin sections at 3-4 microns.
COMPONENTS AND REAGENTS
1. Concentrated Methenamine Solution - 4 oz / 120 ml Solution should be stored in Refrigerator
2. Concentrated Stabilized Silver Nitrate Sol. - 4 oz / 120 ml Solution should be stored in Refrigerator
3. Sodium Borate Solution - 4 oz / 120 ml
4. Chromic Acid Solution - 4 oz / 120 ml
5. Sodium Bisulfate Solution - 4 oz / 120 ml
6. Gold Chloride Solution - 4 oz / 120 ml
7. Sodium Thiosulfate Solution - 4 oz / 120 ml
8. Light Green SF Counterstain - 4 oz / 120 ml
9. Control Slides1 stained and 3 unstained
* Working Methenamine Silver Solution
Add 10 ml of Concentrated Methenamine Solution to 40 ml of Distilled Water. Mix. Add 10 ml of Concentrated Stabilized
Silver Nitrate Solution and shake or stir for 1 minute. Add 2 ml of Sodium Borate Solution and stir for 1 minute. Store in the dark until used.
PROCEDURE
Before starting procedure, prewarm waterbath to 62 degree Celsius.
1. Deparaffinize sections and hydrate to deionized water.
2. Treat sections in Chromic Acid Solution for 1 hour.
3. Rinse sections under tap water.
4. Treat sections in Sodium Bisulfate Solution for 1 minute.
5. Wash sections quickly through two changes of distilled water, 30 seconds each.
6. Treat sections in *Working Methenamine Silver Solution for twenty minutes in prewarmed waterbath.
7. Rinse sections through two changes of distilled water.
8. Treat sections in Gold Chloride Solution for 1 minute.
9. Rinse sections briefly in tap water for 2 minutes.
10. Treat sections in Sodium Thiosulfate Solution for 1 minute.
11. Rinse sections in tap water for 2 minutes.
12. Stain sections in Light Green Counterstain for 30 seconds with agitation. Increasing time will increase intensity.
13. Dehydrate sections through three changes of Reagent Alcohol
14. Clear sections through two changes of Xylene or Xylene substitutes.
15. Coverslip with Clearslip.
RESULTS
Pneumocystis C.-Black to Gray
Fungus-Black to Gray
Mucin-Black to Gray
Basement MembraneBlack to Taupe
BackgroundGreen
NOTES
1. Distilled water is recommended to be used on all the procedure steps. Pending on water source, it is suggested to use deionized or steam distilled water, because natural or processed water qualities in different regions may have an effect on staining results. Always use Distilled Water when manufacturing your Working Methenamine Silver Solution.
2. Working Methenamine Silver Solution should be made fresh before use. Working Methenamine Silver Solution has a shelf life of 4 months without the addition of the Sodium Borate Solution. This solution must be stored in the refrigerator. With the addition of Sodium Borate, the working solution should be stored in the dark and has a shelf life of 2 hours. Black precipitate will develop on the bottom of the incubation jar. This is a normal event.
3. It is strongly suggested to use a waterbath instead of an oven. A waterbath will heat the Working Methanamine Solution more rapidly than your conventional lab oven. If you use an oven, increase your time to 60 minutes.
4. Storing the kit in the refrigerator will not change the efficacy of each non-refrigerated solution. If some undissolved salts appear on the bottom of each bottle, simply heat the solution to room temperature and shake bottle.
REFERENCES
1. AFIP, Manual of Histologic Methods, McGraw Hill Book Co., 1968, pp. 230
2. Molecular Cell Biology, James Darnell, Harvey Lodish, Scientific American Books. 1981
Dev. 8/97
