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

Materials:

• Xylene
• Reagent alcohol histology grade
• Methanol
• 30% Hydrogen Peroxide
• Hydrophobic barrier pen
• Retrieval Buffer-Citrate Buffer pH 6.0
• Retrieval Buffer-Tris-EDTA Buffer pH 9.0
• Wash Solution
• Normal Goat Serum Blocking Solution
• IHC Antibody Diluent
• Goat anti-Rabbit IgG Antibody (Bethyl cat# A120-201P, A120-401P, A120-501P)
• DAB Substrate (KPL DAB Reagent Set #54-10-00)
• IHC Hematoxylin
• IHC Bluing Solution
• Mounting media
• Coverglass

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 perservative.

Wash solution

Secondary Antibody

Goat anti-Rabbit IgG Antibody-horseradish peroxidase conjugated. (Bethyl cat# A120-201P, A120-401P, 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

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
    • Prepare primary antibody with IHC Antibody Diluent
    • Optimal working dilutions should be determined experimentally by the investigator.
    • Refer to Bethyl antibody datasheet for suggested dilution range.
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 development - 5-10 minutes. Monitor development with microscope. Do not develop longer than 10 minutes.
18. Wash Solution - 3 changes for 5 minutes each
19. IHC Hematoxylin - 1-3 minutes
20. Wash Solution - 3 changes for 5 minutes each
21. IHC Bluing Solution - 1-2 minutes
22. dH20 rinse
23. 70% reagent alcohol - 3 minutes
24. 95% reagent alcohol - 2 changes for 3 minutes each
25. 100% reagent alcohol - 3 changes for 3 minutes each
26. Xylene - 3 changes for 3 minutes each
27. Mount and coverslip.
28. Place slides flat to dry.