Immunofluorescence Labelling

Immuno-Labelling Frozen Sections or Cells Grown On Coverslips

  1. Air dry sections/cells onto a polylysine coated slide.

  2. Use a PAP pen to mark around frozen sections (not necessary for cells).

  3. Rehydrate in PBS + 0.02% BSA for 3mins.

  4. Permeabilise with PBS + 0.2% Triton-X.

  5. Wash with PBS + 0.02% BSA for 1min (x2).

  6. Block with serum free proein block or serum from the same species as the secondary antibody for 30mins.

  7. Drain off surplus fluid, do not wash.
  8. Add primary antibody for 45mins.

  9. Wash gently in PBS + 0.02% BSA fro 2mins (x3).

  10. Incubate with secondary antibody (30mins in dark). Everything from this point should be kept in the dark.

  11. Wash in d.H2O then air dry.

  12. Mount in antifade reagent and coverslip.

If using tissue sections requiring fixation then add 4% paraformaldehye or possibly 10% formol saline between steps 3 and 4.

 

Troubleshooting

Sections/Cells Fall Off

  • Wash cells/sections more gently.

  • Make sure slides/coverslips are coated with Laminin or both polylysine + laminin.

  • Reduce the amount of washes but be careful this does not increase background.

  • Ensure sections/cells are allowed to fully dry onto the slide.

 

High Background

  • Have the antibodies been through repeated thaw-refreeze cycles (avoid by aliquoting).

  • Have you used protein blocker and if so is it still within its use by date.

  • Check that secondary antibody alone produces no background as it may be non-specifically binding to other binding sites on your specimen.

  • Check unlabelled sections for autofluorescence which may be contributing to background.

 

No Fluorescence Signal

  • If sections/cells are fixed try a different fixative or a shorter fixation period to prevent cross linking masking the binding sites.

  • Try primary antibody on a positive control to check the antibodies effectiveness (if possible).

  • Try incubating primary antibody overnight at 4°C