Skip Navigation LinksHome » Protocols » ABC Technique using Cultured Cells Grown on Glass Cover Slips
Print this page

Method for ABC Technique using Monoclonal Antibodies on Cultured Cells Grown on Glass Cover Slips

  1. Cells are grown as monolayers on acid-washed, dH2O-rinsed, sterile glass cover slips or slides. It may be necessary in some cases to coat cover slips or slides with poly L-lysine.
  2. Wash attached cells in PBS for 5 minutes.
  3. Fix cells in a fixative eg 1% v/v paraformaldehyde in PBS or Acetone or Zamboni’s (the fixative of choice may depend on the antigen recognised) for 10 minutes.
  4. Wash cells in PBS for 2 x 5 minutes.
  5. Cover slips may be attached to glass slides using suitable adhesive, eg Loctite Glassbond, for convenience.
  6. If required, permeabilise cells using 0.25% v/v Triton in PBS for 20 minutes.
  7. Wash cells in PBS for 2 x 5 minutes.
  8. Cover sections with blocking reagent, eg 10% normal rabbit serum in PBS, for 10 minutes.
  9. Remove excess blocking reagent and replace with primary antiserum pre-diluted in blocking reagent for 60 minutes at 25oC or overnight at 4oC, according to the data sheet.
  10. Rinse in PBS for 2 x 5 minutes.
  11. Remove excess PBS and cover with biotinylated rabbit anti-mouse secondary diluted with blocking reagent for 30 minutes at 25oC.
  12. Rinse in PBS for 2 x 5 minutes.
  13. Remove excess PBS and cover with ABComplex/HRP for 30 minutes at 25oC.
  14. Rinse in PBS for 2 x 5 minutes.
  15. Develop with 3 3’ diaminobenzidine tetrahydrochloride (DAB).
  16. Rinse slides in water.
  17. Counterstain with Haematoxylin (if required).
  18. Dehydrate, clear and mount sections with DPX mountant.

Print this page Print this page
Download this page as PDF document (51 kb)

Get Adobe Reader In order to open PDF-files, you will need Adobe Reader. Click here to download Adobe Reader.