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Confocal Microscopy

Here is a quick guide to our immunostaining protocol for confocal microscopy.

Please note that antibody concentrations and incubation times are listed as a guide and should be determined empirically for the specific cell line and antibody used.

 

  1. Seed cells at ~1:5 split ratio on BioCoat collagen treated chamber slides (or equivalent) and grow for 48 hours. You want the cells to be about 30%-50% confluent so that they are well spread, fairly numerous, but not sloughing off or too crowded.
  2. Wash slide chambers with room temperature buffer (either PBS or TBS) 2 times.
  3. Fix cells with pre-chilled  methanol for 10 minutes at -20oC.
  4. Wash cells with room temperature buffer 3 times.
  5. Block 1 hour to overnight with buffer containing 1% BSA and 5% Normal serum (usually goat, but use whatever your secondary antibody source is).
  6. Primary antibody should be diluted in blocking buffer at 1:1000 and incubated at  room temperature for 1 hour or at 4oC overnight.
  7. Wash 3 times with buffer.
  8. Secondary antibody: 1:500-1:1000 diluted in block and incubated at room temperature for 1 hour.
  9. Wash with buffer, 3 times for 5 minutes.
  10. Mount and coverslip using VectaShield hard mounting medium + DAPI.