Immunostaining of Tissue Culture Cells
Adapted from Harlow and Lane, Antibodies: a laboratory manual (CSHL press).
Growing cells on coverslips
Many cells can simply be grown on coverslips that were sterilized by soaking in 70% ethanol and quickly flamed. For some loosely adhering cells, like 293T, it is neccesary to first soak coverslips O/N at room temperature or 37°C in 0.2% gelatin in H2O. In a recent large batch of stainings it seemed that 239T cells adhered better to coverslips soaked for 2 days than 1 day. For gelatin adherens, a simple option for sterilization is to turn on the UV light for a few hours, and then transfer the plates to the 37°C hood.
Fixing cells in paraformaldehyde
Fixation in a cross-linking agent preserves cell structure better than organic solvents, but may not work for all antibodies. Permeabilization with a nonionic detergent (or organic solvent, but this preserves cell structure less) is required to allow acces of the antibody.
- Wash the coverslip gently in PBS. Briefly dipping in a beaker of PBS is sufficient.
- Fix cells 10 min. at room temp in 4% paraformaldehyde in PBS.
- Wash 2x 5 min. in PBS.
- Permeabilize cells for 10 min. in 0.2% Triton X-100 in PBS.
- Optionally block free aldehyde groups with 50 mM Glycine in PBS.
- Wash 3 x 5 min. in PBS.
Note: Paraformaldehyde fixation is apparently not stable, and will be reversed by long incubations in aqueous solutions, so be quick with the stainings.
All steps are at room temperature.
- Dilute primary antibody in PBS + 1% Tween-20 (PBS-T) + 1% BSA + 10% Serum of the organism the secondary antibody was generated in.
- Incubate with primary antibody for 45 min. One way is to put a 100 µl drop of antibody on a piece of parafilm, and lay the coverslip on this (cell side down).
- Wash 3 x 5 in PBS-T.
- Dilute secondary antibody in PBS + 1% Tween-20 (PBS-T) + 1% BSA + 10% Serum of the organism the secondary antibody was generated in.
- Incubate with secondary antibody for 45 min.
- Wash 3 x 5 in PBS-T. Do a last rinse in regular PBS to wash away the soap scum.
Now mount the slide with Vectashield, with or without Dapi.
Optional Dapi staining
- Before the last washes, add 1µg/ml Dapi in PBS-T to the cells. Incubate for 5 minutes.