2-Step Immunofluorescence Protocol: Formalin-fixed, Paraffin-embedded tissues and cell blocks

Preparation of Reagents

Normal Goat Serum Blocking Solution

• 20 ml normal goat serum (filter sterilize with 0.2 µM filter)
• Volume to 100 ml of PBS or TBS

Store at 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 add a preservative

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-Dylight conjugated. (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4)

Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Protect from light. Optimal working dilutions should be determined experimentally by the investigator.

Procedure

1. Xylene - 3 changes for 5 minutes each
2. 100% reagent alcohol - 3 changes for 5 minutes each
3. dH20 rinse
4. Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
5. Wash Solution - 3 changes for 5 minutes each
6. Normal Goat Serum Blocking Solution - 15 minutes
7. 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.
8. Wash Solution - 3 changes for 5 minutes each
9. Secondary Antibody Incubation: 1 hour room temperature. Protect from light.
10. Wash Solution - 3 changes for 5 minutes each
11. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.