Antigen Retrieval for Frozen Sections

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IHC sample preparation (frozen vs. paraffin-embedded)

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IHC blocking non-specific binding

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IHC Handbook

Antigen Retrieval for Frozen Sections

1% Sodium Dodecyl Sulfate (SDS) in PBS:

SDS -------------------------------------- 1 g

0.01M PBS (pH 7.4) ---------------- 100 ml

Mix to dissolve.

1. Rinse sections three times for 5 min each in PBS.
2. Cover sections with 1% SDS solution and incubate for 5 minutes at RT.
3. Rinse 3 X 5 min each in PBS. (It is important to wash sections well).
4. Proceed with staining as normal.

En Bloc Antigen Retrieval:

1. Fix tissue with buffered 4% PFA. The tissue blocks should be cut to a proper size (e.g. slides 3-5 mm thick).
2. Immerse the tissue blocks in Citrate Buffer at 4 degrees Celsius overnight.
3. Place the tissue blocks at 95-100 degrees Celsius for 3-5 minutes.
4. Immediately place the tissue blocks in cold 30% sucrose in PBS and incubate at 4 degrees Celsius until the blocks sink.
5. Immerse the tissue blocks in an embedding medium and freeze quickly with crushed dry ice.
6. The frozen tissue blocks can now be stored at -80 degrees Celsius and process as normal.

* The above information is intended as a guide. Determining the protocol that best meets your need should be determined for each antigen and antibody.