2-Step Immunoperoxidase Protocol:
Formaldehyde-Fixed Cells and Cytospin Preparations

Required Reagents:
3-4% Paraformaldehyde (freshly prepared) or 10% Neutral Buffered Formalin
Triton-X 100
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:
Wash Solution
5 ml Concentrated IHC Wash Solution (cat# IHC-101e)
995 ml dH20
Store at 40 C, expiration 3 months

10% Triton-X 100 Stock
10 ml Triton-X 100
90 ml dH20

0.25% Triton-X 100
25 µl 10% Triton-X 100
1 ml TBS

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. For cells in chambered microscope slides or cells grown on coverslips: Gently rinse off media with TBS – 3 changes for 1 minute each. For cytospins: Allow to air dry after preparation.
2. Formaldehyde fixation – 10-30 minutes
3. Wash Solution - 3 changes for 5 minutes each
4. Circle cytospin with a hydrophobic barrier pen. Do not allow cells to dry for the remaining procedure.
5. 0.25% Triton-X 100 – 2-10 minutes - Optimal working dilutions and incubation times should be determined experimentally by the investigator.
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: 30 minutes – 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.
9. Wash Solution - 3 changes for 5 minutes each
10. Secondary Antibody Incubation: 30 minutes – room temperature.
11. Wash Solution - 3 changes for 5 minutes each
12. Ready-To-Use IHC Hematoxylin (cat# IHC-101g) – 1-3 minutes
13. Wash Solution - 3 changes for 5 minutes each
14. Ready-To-Use IHC Bluing Solution (cat# IHC-101h) – 1-2 minutes
15. dH20 rinse
16. Remove chamber from microscope slide.
17. 70% EtOH – 3 minutes
18. 95% EtOH – 2 changes for 3 minutes each
19. 100% EtOH – 3 changes for 3 minutes each
20. Xylene - 3 changes for 3 minutes each
21. Mount and coverslip.
22. Place slides flat to dry.

NOTES:
This procedure is suitable for Phospho-specific Antibodies