Bethyl will be closing at 1pm on Friday, December 21st for the Christmas holidays. We will resume normal business hours on Wednesday, January 2nd.

Protocols

 

2-Step Immunoperoxidase Procedure:
Epitope Retrieval Method for Formalin-fixed, Paraffin-embedded tissues and cell blocks

 

 

For antibodies made in Rabbits
(3-5µm paraffin sections on charged (plus) slides)

Required Reagents:

Preparation of Reagents:

10mM Citrate Buffer pH 6.0
2.94 g Tri-sodium Citrate (dihydrate)
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.

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 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 - 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 C- 100 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.
  14. Wash Solution - 3 changes for 5 minutes each
  15. Secondary Antibody Incubation: 1 hour – room temperature
  16. Wash Solution - 3 changes for 5 minutes each
  17. DAB development – 5-10 minutes. Monitor development with microscope. Do not develop longer than 10 minutes.
  18. Wash Solution - 3 changes for 5 minutes each
  19. IHC Hematoxylin – 1-3 minutes
  20. Wash Solution - 3 changes for 5 minutes each
  21. IHC Bluing Solution – 1-2 minutes
  22. dH20 rinse
  23. 70% reagent alcohol – 3 minutes
  24. 95% reagent alcohol – 2 changes for 3 minutes each
  25. 100% reagent alcohol – 3 changes for 3 minutes each
  26. Xylene - 3 changes for 3 minutes each
  27. Mount and coverslip.
  28. Place slides flat to dry.