Centre Optical Instrumentation Laboratory

Antigen Retrieval

Enzymatic Method

1. Place dewaxed slides into ultrapure water at 37°C to warm them.

2. Make up enzyme solution.

(i) Warm another 100ml ultrapure water to 37°C

(ii) Add 0.1g CaCl

(iii) 0.1g alpha-chymotrypsin (type II)

(iv) Adjust pH to 7.8

(v) Warm to 37°C

3. Place warmed slides from ultrapure water into enzyme solution for 20 mins (no longer than 30min)

4. Remove slides and put into cold running tap water to stop the reaction

5. Continue with immuno staining protocol.

Pressure Cooker Method

1. Dissolve 5.88g Tri-Sodium Citrate, and 44ml 0.2M HCL in 1956ml ultrapure water.

2. Adjust pH to 6 with 0.5% NaOH

3. Place solution in pressure cooker and apply heat until boiling, do not secure the lid yet.

4. Transfer the dewaxed slides to the pressure cooker and secure the lid.

5. Cook at full pressure for 3mins (5mins max).

6. Cool the slides.

7. Continue the labelling protocol.

Microwave Method

1. Add 2.94g Tris-Sodium Citrate and 22ml 0.2M HCl to 978ml ultrapure water

2. Adjust to pH 6 with NaOH

3. Place de-waxed slides into microwavable dish and add solution.

4. Heat at full power for 15mins (30mins max).

5. Remove slides and allow to cool.

6. Continue with labelling procedure.