Immuno-Labelling
Immunofluorescence Labelling protocol.
Immuno-Labelling Frozen Sections or Cells Grown On Coverslips
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Air dry sections/cells onto a polylysine coated slide.
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Use a PAP pen to mark around frozen sections (not necessary for cells).
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Rehydrate in PBS + 0.02% BSA for 3mins.
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Permeabilise with PBS + 0.2% Triton-X.
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Wash with PBS + 0.02% BSA for 1min (x2).
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Block with serum free proein block or serum from the same species as the secondary antibody for 30mins.
- Drain off surplus fluid, do not wash.
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Add primary antibody for 45mins.
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Wash gently in PBS + 0.02% BSA fro 2mins (x3).
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Incubate with secondary antibody (30mins in dark). Everything from this point should be kept in the dark.
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Wash in d.H2O then air dry.
- 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
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Wash cells/sections more gently.
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Make sure slides/coverslips are coated with Laminin or both polylysine + laminin.
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Reduce the amount of washes but be careful this does not increase background.
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Ensure sections/cells are allowed to fully dry onto the slide.
High Background
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Have the antibodies been through repeated thaw-refreeze cycles (avoid by aliquoting).
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Have you used protein blocker and if so is it still within its use by date.
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Check that secondary antibody alone produces no background as it may be non-specifically binding to other binding sites on your specimen.
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Check unlabelled sections for autofluorescence which may be contributing to background.
No Fluorescence Signal
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If sections/cells are fixed try a different fixative or a shorter fixation period to prevent cross linking masking the binding sites.
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Try primary antibody on a positive control to check the antibodies effectiveness (if possible).
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Try incubating primary antibody overnight at 4°C