2-Step Immunofluorescence Protocol: Fresh Frozen Tissue Sections

Preparation of Materials

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 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.