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Deparaffinize slides a (after drying thoroughly overnight at RT) in 2 changes of xylene (or xylene substitute) for 10 mins each. |
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Transfer slides to 100% alcohol, 2 changes for at least 2 min each. |
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Block endogenous peroxidase activity by incubating for 10 min in 3% H2O2. |
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Rinse 2-3 times in water. |
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Place slides in a plastic coplin jar or Tek® staining dish and fill container with 10 mM citrate bufferb, c , pH 6.0. |
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Place staining dish in microwave with inverted lid on top. If using a probe, set temperature to 193°F. (If your microwave does not have a probe, see reference below.) |
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Mix the solution with a disposable pipet after temperature has been reached and hold that temperature in the microwave for 10 min. |
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Remove slides from microwave and set the covered dish on the counter for an additional 20 min. |
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9. |
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Rinse slides 2-3 times in PBS and continue with Step 10 of "Preparation of Paraffin Sections" |
NOTES:
a. For best adherence, use Superfrost Plus® slides (Poly-L-lysine or silane slides are also acceptable) and use only deionized water in the waterbath when cutting paraffin sections.
b. When staining paraffin-embedded sections, antigens are affected differently by the various methods of pretreatment (citrate buffer, trypsin, or no pretreatment). Please call Technical Service for specific information about antigen retrieval. It should be noted that certain antigenic specificities cannot be detected in paraffin-embedded sections.
c. Preparation of 10 mM Citrate Buffer (pH 6.0)
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Stock A Citrate Buffer |
Stock B Citrate Buffer |
Working 10 mM Citrate Buffer |
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4.2 g Citric Acid |
14.7 g Sodium Citrate |
18 ml Stock A |
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20 mls ddH20 |
500 mls ddH20 |
82 ml Stock B |
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Fill to 1 liter with ddH20 and pH to 6.0 |
Reference:
1. Tacha, DE, Chen T. Modified antigen retrieval procedure: calibration techniques for microwave ovens. Histotechnology 17(4):365,1994.
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