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ICC Cells Coverglass

Basic ICC Protocol - coverslips

  1. Remove culture media from dishes and replace with 2ml PBS.
  2. Replace PBS with 2ml of 2% Fixative and fix for 10min at RT.
  3. Transfer all coverslips to a submerged (in PBS) Coors Holder. Place jar on rotating platform and agitate gently for 10min. Repeat wash twice more in fresh PBS for 10min each.
  4. Transfer holder to ice-cold methanol for 3 min at –20°C, if permeabilization is required. (Or use a mild detergent (0.1 – 0.3% Triton)  Permeabilization is not recommended for plasma membrane proteins.)
  5. Transfer holder to PBS and wash samples in PBS for 3x10minwith agitation. (Prepare antibodies in 10% serum from the host of the secondary antibody)
  6. Transfer coverslips, one at a time, to incubation box and add 250µl of appropriate antibody or PBS/10% serum (controls).
  7. Place the lid on the box and incubate in primary antibodies overnight at 4°C. 
  8. Remove one coverglass from box, and blot excess antibody using a paper towel. Dip coverslip in a beaker of PBS for 5s, then place it into a Coors holder in PBS.
  9. Wash samples in PBS for 3x10min with agitation. (Prepare secondary antibodies in 10% serum)
  10. Transfer coverslips, one at a time, to incubation box and add 250µl of appropriate secondary antibody or PBS/10% serum.
  11. Incubate in appropriate secondary antibodies  for 1hr. at RT.
  12. Remove one coverglass from box, and blot excess antibody using a paper towel.Dip coverslip in a beaker of PBS for 5s, then place it into a Coors holder in PBS.
  13. Wash samples in PBS for 3x10min with agitation.
  14. Transfer coverslips, one at a time, to incubation box and add 250µl of 300nM DAPI. Start a timer on the first coverslip.  Incubate each coverslip for 2min.
  15. One coverglass at a time, allow DAPI to run off onto a paper towel, then place coverglass back into Coors Holder in PBS.  Rinse for 2min with agitation.
  16. Mount coverslips using MOWIOL
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