2-Step Immunoperoxidase Procedure: Epitope Retrieval Method for Formalin-fixed, Paraffin-embedded tissues and cell blocks

Preparation of Materials

10mM Citrate Buffer pH 6.0

2.94 g Tri-sodium Citrate (dihydrate)

Volume to 1 Liter with distilled water

Check pH (pH is usually at 6.0).

Store at room temperature for 3 months or at 4° C for 6 months.

Tris-EDTA Buffer pH9.0 (10mM Tris Base, 1mM EDTA)

1.21 g Tris Base

0.37 g EDTA

Volume to 1 Liter with distilled water

Check pH (pH is usually at 6.0).

Store at room temperature for 3 months or at 4° C for 6 months.

Methanol/H2O2 Peroxidase quench solution

6ml 30% Hydrogen peroxide

194ml Methanol. Use caution when handling and disposing methanol.

Prepare just prior to use.

Normal Goat Serum Blocking Solution

20 ml normal goat serum (Filter sterilize with 0.2 µM filter)

Volume to 100 ml with PBS or TBS.

Store 2-8 C, discard after 3 months.

IHC Antibody Diluent (50mM TBS, 1% BSA)

190 ml 1X TBS

2 g BSA (Sigma A9647)

Volume to 200 ml with TBS.

Store 2-8 C, discard after 1 year.

*May want to use a preservative.

Wash solution

Can be any of the following: PBS, TBS or 0.01% Tween 20 in dH20. TBS wash solution is recommended for phospho antibodies.

Secondary Antibody

Goat anti-Rabbit IgG Antibody-horseradish peroxidase conjugated. Bethyl cat# HRP cross-absorbed (A120-201P), HRP human-absorbed (A120-401P), HRP highly cross-absorbed (A120-501P)

Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Optimal working dilutions should be determined experimentally by the investigator.

DAB Solution

DAB Reagent set (KPL Cat #54-10-00)

IHC Hematoxylin

10 ml Gill 1-regular strength (StatLab #SL97-16)

90 ml distilled water

IHC Bluing Solution

50ml 10X Tris pH 7-8

450 ml distilled water

Procedure

1. Xylene - 3 changes for 5 minutes each
2. 100% Reagent alcohol - 3 changes for 5 minutes each
3. Methanol/H2O2 - 5-15 minutes
4. dH20 rinse
5. Place prepared Epitope Retrieval Buffer (refer to Bethyl antibody datasheet for appropriate epitope retrieval buffer) into steamer or water bath and heat to 96-100%deg; C
6. Add rack of slides to hot Epitope Retrieval Buffer - 20 minutes
7. Remove container and slides from steamer or water bath and cool on bench - 20 minutes
8. Slowly add dH20 for 5 minutes
9. dH20 rinse - 1 minute
10. Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
11. Wash Solution - 3 changes for 5 minutes each
12. Normal Goat Serum Blocking Solution - 15 minutes
13. Primary Antibody Incubation: 1 hour - room temperature
14. Prepare primary antibody with IHC Antibody Diluent
15. Optimal working dilutions should be determined experimentally by the investigator.
16. Refer to Bethyl antibody datasheet for suggested dilution range.
17. Wash Solution - 3 changes for 5 minutes each
18. Secondary Antibody Incubation: 1 hour - room temperature
19. Wash Solution - 3 changes for 5 minutes each
20. DAB development - 5-10 minutes. Monitor development with microscope. Do not develop longer than 10 minutes.
21. Wash Solution - 3 changes for 5 minutes each
22. IHC Hematoxylin - 1-3 minutes
23. Wash Solution - 3 changes for 5 minutes each
24. IHC Bluing Solution - 1-2 minutes
25. dH20 rinse
26. 70% reagent alcohol - 3 minutes
27. 95% reagent alcohol - 2 changes for 3 minutes each
28. 100% reagent alcohol - 3 changes for 3 minutes each
29. Xylene - 3 changes for 3 minutes each
30. Mount and coverslip.
31. Place slides flat to dry.