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 Immunofluorescence Protocol:
Fresh Frozen Tissue Sections

 

 

For antibodies made in Rabbits
(5-15 µm cryosections on charged (plus) slides)

 

Required Reagents:

Preparation of Reagents:

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 FITC. (Bethyl cat# A120-201F) Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Optimal working dilutions should be determined experimentally by the investigator.

Procedure:

  1. Allow cryosections to air dry 30 minutes to 1 hour prior to fixation.
  2. Acetone 10 minutes
  3. Air dry 30 minutes in hood
  4. Circle section with a hydrophobic barrier pen.
  5. Wash Solution - 3 changes for 5 minutes each. Do not allow sections to dry for the remaining procedure.
  6. IHC 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.
  8. Wash Solution - 3 changes for 5 minutes each
  9. Secondary Antibody Incubation: 30 minutes room temperature. Protect from light.
  10. Wash Solution - 3 changes for 5 minutes each
  11. dH20 rinse
  12. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.