Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

Basic outline protocol for immunohistochemistry on unfixed cytostat-thin tissue sections

method/1/Ki67 photo.jpg


50 ml of 1xPBS pH 7.4 50ml of 1% BSA/PBS Primary antibody: As recommended by manufacturer Secondary antibody: As recommended by manufacturer – Alexa dyes usually work best at 1:400


  • Cryostat sections are picked up on subbed slides (chrome-alum gelatin coated) and air dried for at least 2 hours.

Alternatively Superfrost or Poly-L-Lysine coated slides can be used.

  • Draw a circle around section with hydrophobic pen.
  • Wash sections with PBS 3 times
  • Incubate twice in 100% Ethanol for 2 minutes each.

Incubation for 20 min in PBS + 4% formaldehyde can be used as an alternative fixative.

  • Block with BSA/PBS for 20 minutes.
  • Add 1o antibody for 1 hour.
  • Wash with BSA/PBS 3 times.
  • Add 2o Ab for 1hr
  • Wash with BSA/PBS 3 times
  • Wash with PBS 3 times.
  • A counterstain (e.g. DAPI or PI) may be added for a few minutes, followed by PBS washing.
  • Mount with aqueous based fluorescent mounting medium (mowiol / prolong gold), dry edges, seal with nail polish.

This method is based, with permission, on an original protocol available here.