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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.

Staining cells

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.