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

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# 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

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

• 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.

1. Wash Solution - 3 changes for 5 minutes each
2. Secondary Antibody Incubation: 1 hour - room temperature
3. Wash Solution - 3 changes for 5 minutes each
4. DAB development - 5-10 minutes. Monitor development with microscope. Do not develop longer than 10 minutes.
5. Wash Solution - 3 changes for 5 minutes each
6. IHC Hematoxylin - 1-3 minutes
7. Wash Solution - 3 changes for 5 minutes each
8. IHC Bluing Solution - 1-2 minutes
9. dH20 rinse
10. 70% reagent alcohol - 3 minutes
11. 95% reagent alcohol - 2 changes for 3 minutes each
12. 100% reagent alcohol - 3 changes for 3 minutes each
13. Xylene - 3 changes for 3 minutes each
14. Mount and coverslip.
15. Place slides flat to dry.