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

This 2-step immunoperoxidase protocol should be used for cells grown in culture and cytospin preparations. Includes the required reagents, preparation of reagents and the step-by-step procedure outline.

For antibodies made in Rabbits

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

Materials:

  • Acetone
  • Hydrophobic barrier pen
  • 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:

Norml 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

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.
  8. Wash solution- 3 changes for 5 minutes each
  9. Secondary Antibody Incubation: 30 minutes room temperature
  10. Wash Solution - 3 changes for 5 minutes each
  11. DAB development - 5-10 minutes. Monitor development with microscope. Do not develop longer than 10 minutes
  12. Wash Solution - 3 changes for 5 minutes each
  13. IHC Hematoxylin - 1-3 minutes
  14. dH20 rinse- 2 changes for 3 minutes each
  15. Bluing Solution - 1-2 minutes
  16. dH20 rinse
  17. 70% reagent alcohol - 3 minutes
  18. 95% reagent alcohol - 2 changes for 3 minutes each
  19. 100% reagent alcohol - 3 changes for 3 minutes each
  20. Xylene - 3 changes for 3 minutes each
  21. Mount and coverslip.
  22. Place slides flat to dry.
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