Antigen Retrieval
Formalin fixation forms protein cross-links that can mask the antigenic sites in tissue specimens, thereby creating weak or false negative staining for immunohistochemical detection of certain proteins. There are currently several antigen retrieval methods commonly used and the protocols below are only intended as a general guide.
Heat Induced Epitope Retrieval (HIER)
| Citric acid (anhydrous) | 1.92 g |
| Distilled water | 1000 ml |
| Mix to dissolve. Adjust pH to 6.0 w/ 1N NaOH and add 0.5 ml of Tween 20, mix well. | |
Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or CitrateBuffer until temperature reaches 95-100 degrees Celsius. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minute. Remove the staining dish to room temperature and allow the slides to cool for 20 minutes before proceeding with normal staining procedure.
| Tris | 6.1 g |
| Sodium Chloride | 8.8 g |
| Distilled water | 1000 ml |
| Mix to dissolve. Adjust pH to 9.0 using concentrated HCl and then add 0.5 ml of Tween 20 and mix well. | |
Pre-heat steamer or water bath with staining dish containing TBS until temperature reaches 95-100 degrees Celsius. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minute. Remove the staining dish to room temperature and allow the slides to cool for 20 minutes before proceeding with normal staining procedure.
Proteolytic Induced Epitope Retrieval (PIER)
| Trypsin stock solution (0.5%) | 1 ml |
| Calcium chloride stock solution 1% | 1 ml |
| Distilled Water | 8 ml |
| Adjust pH to 7.8 with 1N NaOH. | |
Cover sections with trypsin working solution and incubate for 10-20 minutes at 37 degrees Celsius in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user). Allow sections to cool at room temperature for 10 minutes.
| Proteinase K Stock Solution (20X) | 1 ml |
| TE Buffer, pH8.0 | 19 ml |
| Mix well. | |
Cover sections with ProteinaseK working solution and incubate for 10-20 minutes at 37 degrees Celsius in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user). Allow sections to cool at room temperature for 10 minutes.
Antigen Retrieval for Frozen Sections
| SDS |
1 g |
| 0.01M PBS (pH 7.4) |
100 ml |
| Mix to dissolve. | |
Rinse sections three times for 5 min each in PBS. Cover sections with 1% SDS solution and incubate for 5 minutes at room temperature. Rinse 3 X 5 min each in PBS. (It is important to wash sections well). Proceed with staining as normal.
En Bloc Antigen Retrieval
- Fix tissue with buffered 4% PFA. The tissue blocks should be cut to a proper size (e.g., slices 3–5 mm thick).
- Immerse the tissue blocks in Citrate buffer at 4C overnight.
- Place the tissue blocks at 95-100?C 3–5 min.
- Immediately place the tissue blocks in cold 30% sucrose in PBS and incubate at 4 C until the blocks sink.
- Immerse the tissue blocks in an embedding medium and freeze quickly with crushed dry ice. The frozen tissue blocks can now be stored at -80C and process as normal.
* This information is only intended as a general guide. Always observe safe laboratory procedures and use appropriate safety equipment