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

(3-5µm paraffin sections on charged (plus) slides)

Bethyl Laboratories IHC Reagents are strongly recommended for best results.

Required Reagents:
Xylene
Ethanol- histology grade
Methanol
30% Hydrogen Peroxide
Hydrophobic barrier pen
Concentrated Epitope Retrieval Buffer-Reduced pH (Bethyl cat# IHC-101a)
Concentrated IHC Wash Solution (Bethyl cat# IHC-101e)
Ready-To-Use IHC Blocking Solution (Bethyl cat# IHC-101b)
Ready-To-Use IHC Antibody Diluent (Bethyl cat# IHC-101c)
Concentrated anti-Rabbit IHC Antibody (Bethyl cat# IHC-101d)
Concentrated DAB Substrate (Bethyl cat# IHC-101f)
Ready-To-Use IHC Hematoxylin (Bethyl cat# IHC-101g)
Ready-To-Use IHC Bluing Solution (Bethyl cat# IHC-101h)
Mounting media
Coverglass

Preparation of Reagents:
Methanol/ H2O2
6 ml 30% Hydrogen Peroxide
194 ml Methanol
Prepare just prior to use.

Epitope Retrieval Buffer
4 ml Concentrated Epitope Retrieval Buffer-Reduced pH (Bethyl cat# IHC-101a)
196 ml dH20
Adjust pH 6.0 if necessary

Wash Solution
5 ml Concentrated IHC Wash Solution (cat# IHC-101e)
995 ml dH20
Store at 4 C, expiration 3 months

Secondary Antibody
1 drop Concentrated anti-Rabbit IHC Antibody (cat# IHC-101d)
2 ml Ready-To-Use Antibody Diluent (cat# IHC-101c)
Prepare just prior to use.

DAB Solution
2.5 ml dH20
Concentrated DAB Substrate (cat# IHC-101f):
1 drop DAB Solution A
1 drop DAB Solution B
1 drop DAB Solution C
Prepare just prior to use.

Procedure:

1. Xylene - 3 changes for 5 minutes each
2. 100% EtOH - 3 changes for 5 minutes each
3. Methanol/H2O2 - 5-15 minutes
4. dH20 rinse
5. Place prepared Epitope Retrieval Buffer into steamer or water bath and heat to 96 C- 100 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. Ready-To-Use IHC Blocking Solution (cat# IHC-101b) - 15 minutes
13. Primary Antibody Incubation: 1 hour – room temperature
    1. Prepare primary antibody with Ready-To-Use IHC Antibody Diluent (Bethyl cat# IHC-101c)
       Optimal working dilutions should be determined experimentally by the investigator.
       For Bethyl IHC Antibodies: 1:100 – 1:500
14. Wash Solution - 3 changes for 5 minutes each
15. Secondary Antibody Incubation: 1 hour – room temperature
16. Wash Solution - 3 changes for 5 minutes each
17. DAB Solution – 5-10 minutes (monitor development with microscope)
18. Wash Solution - 3 changes for 5 minutes each
19. Ready-To-Use IHC Hematoxylin (cat# IHC-101g) – 1-3 minutes
20. Wash Solution - 3 changes for 5 minutes each
21. Ready-To-Use IHC Bluing Solution (cat# IHC-101h) – 1-2 minutes
22. dH20 rinse
23. 70% EtOH – 3 minutes
24. 95% EtOH – 2 changes for 3 minutes each
25. 100% EtOH – 3 changes for 3 minutes each
26. Xylene - 3 changes for 3 minutes each
27. Mount and coverslip.
28. Place slides flat to dry.