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

Preparation of Reagents

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 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.

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

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. 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 for 1-2 minutes 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. Protect from light.
11. Wash Solution - 3 changes for 5 minutes each
12. Remove chamber from microscope slide.
13. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.