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2-Step Immunoperoxidase Protocol: Formaldehyde-Fixed Cells and Cytospin Preparations

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

For antibodies made in Rabbits

Materials:

  • 3-4% Paraformaldehyde (freshly prepared) or 10% Neutral Buffered Formalin
  • Triton-X 100
  • 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 Reagents:

Wash Solution

PBS or TBS. TBS wash solution is recommended for phospho antibodies.


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.


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


10% Triton-X 100 Stock

10 ml Triton-X 100
90 ml dH20


0.25% Triton-X 100

25 µl 10% Triton-X 100
1 ml TBS


Procedure:

  1. For cells in chambered microscope slides or cells grown on coverslips: Gently rinse off media with TBS - 3 changes for 1 minute each. For cytospins: Allow to air dry after preparation.
  2. Formaldehyde fixation - 10-30 minutes
  3. Wash Solution - 3 changes for 5 minutes each
  4. Circle cytospin with a hydrophobic barrier pen. Do not allow cells to dry for the remaining procedure.
  5. Permeabilization step: 0.25% Triton-X 100 - 2-10 minutes. Optimal working dilution and incubation time should be determined experimentally by the investigator.
  6. Wash Solution - 3 changes for 5 minutes each
  7. Normal goat serum Blocking Solution - 15 minutes
  8. Primary Antibody Incubation: 30 minutes - room temperature. Prepare primary antibody with IHC Antibody Diluent. Optimal working dilutions should be determined experimentally by the investigator.
  9. Wash Solution - 3 changes for 5 minutes each
  10. Secondary Antibody Incubation: 30 minutes - 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. Remove chamber from microscope slide.
  19. 70% reagent alcohol - 3 minutes
  20. 95% reagent alcohol; 2 changes for 3 minutes each
  21. 100% reagent alcohol; 3 changes for 3 minutes each
  22. Xylene - 3 changes for 3 minutes each
  23. Mount and coverslip.
  24. Place slides flat to dry.

NOTE: This procedure is suitable for Phospho-specific Antibodies

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