Antigen Retrieval

Protocol for Antigen Retrieval.

Enzymatic Method

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

• Make up enzyme solution.

  • Warm another 100ml ultrapure water to 37°C

  • Add 0.1g CaCl

  • Add 0.1g alpha-chymotrypsin (type II)

  • Adjust pH to 7.8

  • Warm to 37°C

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

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

• Continue with immuno staining protocol.

Pressure Cooker Method

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

• Adjust pH to 6 with 0.5% NaOH

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

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

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

• Cool the slides.

• Continue the labelling protocol

Microwave Method

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

• Adjust to pH 6 with NaOH

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

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

• Remove slides and allow to cool.

• Continue with labelling procedure.