Make sure you perform this procedure BEFORE THE CELLS BECOME CONFLUENT. If they are confluent, they'll sheet off the coverslips and then you won't be able to stain them.
- Aspirate media leaving cells (be careful not to aspirate any of the cells)
- Gently add 500 uL of Fixation buffer (drizzled down the sides of the wells). Wait for 20 minutes at room temperature.
- Use the pipette to aspirate the Fixation Buffer. DO NOT ASPIRATE WITH THE VACUUM; the fixation buffer contains paraformaldehyde, which must be treated as hazardous waste. Instead transfer the fixation buffer to the formaldehyde waste container.
- Add 500 uL PBS; transfer the 24-well plate to the benchtop.
- Squirt some water on the bench, then lay down several sheets of parafilm side-by-side. In the steps below, pipette the solution onto a spot on the parafilm (it will form a "bubble"), then float the coverslip with the cells upside-down on the solution. When you're done, aspirate the liquid off of the parafilm.
- CAREFULLY fish the coverslips out of the wells. Wash the coverslips with 200 µl PBS for 5 minutes.
- Permeabilize the cells with 100 µl 0.2% Triton X-100 for 15 minutes.
- Block the cells with 200 µl PBS+4% BSA for at least 20 minutes (longer is better)
- Prepare 25 µl primary antibody solution for each coverslip: PBS + 4% BSA + antibodies
- Incubate cells with 25µl primary antibody solution for 60 minutes. Be careful that they don't dry out! Saturate a kimwipe or paper towel with water, then put the the wipe in a cover over the incubating coverslips. (The top of a cryobox works well.)
- Wash the coverslips 3x: 200 µl PBS, 5 minutes each.
- Prepare 25 µl secondary antibody solution: PBS + 4% BSA + 2° antibody + DAPI (diluted 1:500, ~1 ug/ml)
- Incubate the cells as above (30 minutes); this time, make the cover light-tight (again, use the top of a cryobox.)
- Wash the coverslips 3x: 200 µl PBS, 5 minutes each.
- Pipette 20 µl of embedding solution on a microscope slide. Slowly lower the sample onto it, taking care to avoid bubbles!
- Store the slide flat, in the dark, overnight to let the embedding solution cure.