Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom
Basic outline protocol for immunohistochemistry on unfixed cytostat-thin tissue sections
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.