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His Question: What is the recommended protocol and in particular the antigen retrieval method for the attached p16 antibody.
Asked on Nov 01 2012
Thank you for contacing us. I have placed the protocol below, please let me know if you have any further questions.
2.1.1. Heat slides in an oven at 65 °C for 1 hour.
2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (5 min each), followed by two 100% ethanol rinses (5 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by H2O and a TBST wash for 5 min on a shaker.
2.2. Antigen Retrieval
2.2.1 Immerse slides into staining dish containing Antigen Retrieval Solution (citrate pH 6 was used)
2.2.2 Place covered staining dish into the rice cooker. Add 120 mL dH2O and press “cook”.
2.2.3 When “cook” is turned to “warm” (about 20–30 min), unplug the cooker and remove the staining dish to the bench top.
2.2.4 Allow to cool down, without cover, for 20 min.
2.3.1. Wash slides with TBST for 5 min on a shaker.
2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 10 min.
2.3.3. Wash slides three times with TBST (3 min each on a shaker).
2.3.4. Block slides with the blocking solution for 1 hour.
2.3.5. Dilute primary antibody in the blocking buffer per recommendation on the data sheet.
2.3.6. Apply primary antibody to each section 1/10 dilution and incubate overnight in the humidified chamber (4 ºC).
2.3.7. Wash slides three times with TBST (3 min each on a shaker).
2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer’s recommendation; incubate for 1 hour at room temperature.
2.3.9. Wash slides three times with TBST (3 min each on a shaker).
2.3.10. Add freshly prepared DAB substrate to the sections.
2.3.11. Incubate tissue sections with the substrate at room temperature until suitable staining develops (generally 2–5 min).
2.3.12. Rinse sections with water.
2.3.13. Counterstain with Hematoxylin.
2.3.14. Rinse sections with water.
2.3.15. Dehydrate samples using two rinses with 100% Ethanol (20 dips per rinse) followed by two rinses with Xylene (30 dips per rinse).
2.3.16. Mount coverslips on slides using Permount medium.
Answered on Nov 01 2012