Photograph of scientist pipetting solutions in a lab.  

This protocol provides general instructions for fixation, permeabilization, and blocking to prepare your cells for immunolabeling. Fixing and permeabilizing cells generally locks them in place and makes it possible for larger molecules such as antibodies to access the interior of the cell.

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Reminder
Planning on using a mountant during your experiment? See vessel considerations and mounting coverslips
Protocol tip
If you are running short on time and have a robust system, you can block for 30 minutes instead of 60

What you need

  • Live cells
  • Phosphate-buffered saline (PBS)
  • 4% Paraformaldehyde
  • 0.5% Triton® X-100
  • 3% w/v bovine serum albumin in PBS


General fix, perm & block protocol

The volumes given in this protocol are good for a single well of adherent cells grown in a 6-well plate or a 35 mm dish.

1 Remove the medium from your cells (tip the vessel and pipet from the side).
2 Add 1 mL of 4% (w/v) formaldehyde solution in phosphate-buffered saline (PBS).
3 Incubate for 15 minutes at room temperature for most targets.
4 Remove the fixative solution and wash by pipetting PBS (a volume that will just cover the cells) against the side of the vessel, gently swishing the solution from side to side, and then tilting the vessel and removing the PBS; repeat 3 times. The fixed sample can be stored for several days at 5°C if needed. Learn how to wash 
5 Add 1 mL of 0.5% Triton® X-100 (v/v) in PBS.
6 Incubate for 15 minutes at room temperature.
7 Remove the permeabilization solution and wash 3 times with PBS as in step 4. 
Learn how to wash 
8 Add 2 mL of 3% bovine serum albumin in PBS (or a different  blocking solution if required).
9 Incubate for at least 60 minutes (up to overnight) at room temperature.
10 Go to cell staining or immunolabeling.
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