De-Paraffinization and Antigen Retrieval
1. Incubate slide at 60°C for 60 min.
2. Bath the slides in antigen retrieval solution and heat it to boil with a microwave on medium power.
Once boiling, immediately transfer to a small power and keep faint boiling while heating for 2-3 min.
3. Cool them to room temperature and wash rocking in distilled water several times.
4. Immerse slides in 3% H2O2 (in fresh methanol) for 15 min at room temperature.
5. Wash with TBS (pH 7.6) three times, 5 min each time.
Staining with Primary Antibody
1. Dilute primary antibody with 3% BSA in TBS. Cover the tissue section on the slide with diluted primary
antibody (use 50 – 150μl for each slide).
2. Incubate at 37°Cfor 2 hours (The optimal incubation time, incubation temperature, and antibody dilution
should be determined by the individual laboratory).
3. Wash with TBS three times, 5 min each time.
Staining with Secondary Antibody
1. Incubate with 100-200μl Polymer Enhancer. Incubate 30 min at room temperature.
2. Wash with TBS for 3 times, 5 min each time.
3. Incubate with 100-200μl Polymerized HRP and incubate 30 min at 37°C.
4. Wash with TBS for 3 times, 5 min each time.
5. Add DAB solution and incubate 3-5 min(The reaction progress and the optimal time should be determine
according to microscope).
6. Wash with distilled water for 2 times, 5 min each time.
7. Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl-
ethanol for 1-10 seconds, wash with distilled water. Doing a counterstain of sections in hematoxylin for
1-2 min and wash them with distilled water.
8. Dehydrate through 100% ethanol for 5 min, 90% ethanol for 5 min, 75% ethanol for 5 min, and coverslip
with mounting medium.