2-Step Immunoperoxidase Protocol: Cells Grown in Culture and Cytospin Preparations

Preparation of Materials

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. For cells in chambered microscope slides or cells grown on coverslips: Gently rinse off media with PBS 3 changes for 1 minute each. For cytospins: Allow to air dry after preparation.
2. Acetone fixation 10 minutes
3. Air dry 30 minutes in hood
4. Circle cytospin with a hydrophobic barrier pen.
5. Wash solution. Do not allow sections to dry for the remaining procedure.
6. Normal Goat Serum Blocking Solution - 15 minutes
7. Primary Antibody Incubation: 30 minutes 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: 30 minutes 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. dH20 rinse- 2 changes for 3 minutes each
8. 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.