2-Step Immunoperoxidase Procedure:
Formalin-fixed, Paraffin-embedded tissues and cell blocks

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

Required Reagents:
Xylene
Ethanol- histology grade
Methanol
30% Hydrogen Peroxide
Hydrophobic barrier pen
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.

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 – 1 minute
5. Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
6. Wash Solution - 3 changes for 5 minutes each
7. Ready-To-Use IHC Blocking Solution (cat# IHC-101b) - 15 minutes
8. Primary Antibody Incubation: 1 hour – room temperature. 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
9. Wash Solution - 3 changes for 5 minutes each
10. Secondary Antibody Incubation: 1 hour – room temperature
11. Wash Solution - 3 changes for 5 minutes each
12. DAB Solution – 5-10 minutes (monitor development with microscope)
13. Wash Solution - 3 changes for 5 minutes each
14. Ready-To-Use IHC Hematoxylin (cat# IHC-101g) – 1-3 minutes
15. Wash Solution - 3 changes for 5 minutes each
16. Ready-To-Use IHC Bluing Solution (cat# IHC-101h) – 1-2 minutes
17. dH20 rinse
18. 70% EtOH – 3 minutes
19. 95% EtOH – 2 changes for 3 minutes each
20. 100% EtOH – 3 changes for 3 minutes each
21. Xylene - 3 changes for 3 minutes each
22. Mount and coverslip.
23. Place slides flat to dry.