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2-Step Immunoperoxidase Procedure: Formalin-fixed, Paraffin-embedded tissues and cell blocks

See the protocol steps detailing the 2-step immunoperoxidase procedure: formalin-fixed, paraffin-embedded tissues and cell blocks method, with the reagents and procedure to successfully complete this experiment.

For antibodies made in Rabbits

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

Materials:

  • Xylene
  • Reagent alcohol histology grade
  • Methanol
  • 30% Hydrogen Peroxide
  • 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:

Methanol/H2O2

6 ml 30% Hydrogen Peroxide
194 ml 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

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 - 15 minutes
  4. dH20 rinse – 1 minute
  5. Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
  6. Wash Solution - 3 changes for 5 minutes each
  7. Normal Goat Serum Blocking Solution - 15 minutes
  8. 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.
  9. Wash Solution - 3 changes for 5 minutes each
  10. Secondary Antibody Incubation: 1 hour - room temperature
  11. Wash Solution - 3 changes for 5 minutes each
  12. DAB development - 5-10 minutes. Monitor development with microscope. Do not develop longer than 10 minutes.
  13. Wash Solution - 3 changes for 5 minutes each
  14. IHC Hematoxylin - 1-3 minutes
  15. Wash Solution - 3 changes for 5 minutes each
  16. IHC Bluing Solution - 1-2 minutes
  17. dH20 rinse
  18. 70% reagent alcohol - 3 minutes
  19. 95% reagent alcohol - 2 changes for 3 minutes each
  20. 100% reagent alcohol - 3 changes for 3 minutes each
  21. Xylene - 3 changes for 3 minutes each
  22. Mount and coverslip.
  23. Place slides flat to dry.
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