Retrievals

Immunohistochemistry

The Immunohistochemistry staff furnishes the protocols for heat-induced epitope retrieval and enzymatic epitope retrieval.

Heat-Induced Epitope Retrieval

Decloaker

1. Add 500ml distilled water to the pan of the Decloaker™.
2. Place a full rack of slides in a Tissue Tek™ container containing 250ml of retrieval solution.
3. Decloak for 5 minutes.
4. Depressurize for 10 minutes.
5. Remove pan top and cool for 10 minutes.
6. Rinse in distilled water for 3 minutes. Repeat twice.

Equipment information: Decloaker (http://www.niehs.nih.gov/research/atniehs/labs/lep/path-support/immuno/equipment.cfm#decloaker)

Microwave (Panasonic Model No. NN-S744WF 1200 Watts)

1. Place a full rack of slides in a Tissue Tek™ container containing 250ml of retrieval solution.
2. Microwave for 5 minutes at level 5.
3. Cool for 1 minute (Add 50ml of retrieval solution to the container, if necessary).
4. Microwave again for 5 minutes at level 5.
5. Remove the slides from the microwave oven and cool 20 minutes at room temperature.
6. Rinse slides in distilled water for 2 minutes. Repeat twice.

Equipment information: Microwave (http://www.niehs.nih.gov/research/atniehs/labs/lep/path-support/immuno/equipment.cfm#microwave)

Steamer

1. Place two containers in the steamer basket.
2. Fill container 1 with 250ml of 1X Automation Buffer.
3. Fill container 2 with 250ml of retrieval solution.
4. Turn steamer dial to 35 minutes.
5. Place slides in container 1 for 5 minutes.
6. Remove slides, drain and transfer to container 2 for 30 minutes.
7. Remove slides from the steamer and cool at room temperature for 20 minutes.
8. Rinse slides in distilled water for 1 minute to stop the reaction.
9. Incubate slides in 1X Automation Buffer for 5 minutes.
10. Proceed with stain.

Equipment information: Steamer (http://www.niehs.nih.gov/research/atniehs/labs/lep/path-support/immuno/equipment.cfm#steamer)

Enzymatic Epitope Retrieval

Pepsin

1. Incubate slides in 1X Automation Buffer at 37°C.
2. Remove buffer and apply ready-to-use Carezyme-II solution for 5 minutes at 37°C.
3. Rinse slides in distilled water for 1 minute to stop digestion.

Pronase

Pronase is purchased concentrated from DakoCytomation (http://www.dako.com/). To prepare, aliquot pronase in 100μl volumes and store at -20°C.

1. Dilute 100µl stock with
2. 9ml of 1X Automation Buffer.
3. Apply the 0.05% pronase solution for 5 minutes at room temperature.
4. Rinse in distilled water for 1 minute to stop digestion.

(For Vimentin staining on mouse tissue, the pronase treatment period is 30 minutes.)

Trypsin

Traditionally, enzyme retrieval with trypsin works at a concentration of 0.01% and is conducted at 37°C. The incubation time can vary and is dependent on the tissue type and the sensitivity of the antibody. For development, the tissue is tested at 5-minute intervals, so multiple sections (slides) are required. One important note, trypsin looses 75% of its reactivity within 30 minutes of preparation. If trypsin is the method of choice for retrieval, the concentration and incubation time will be listed for the respective protocol.

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