Immunofluorescence
IF
Immunofluoresence (IF) is an assay typically used to detect antigens, via fluorescently-tagged antibodies (labelled with fluorophores or fluorochromes), in biological samples. It can be used to analyse the distribution of proteins, glycans and other small or non-biological molecules – the fluorophore allows visualisation of the target under a fluorescent microscope.
IF can be direct, or indirect: (i) direct IF uses a single antibody specific to the target of interest, and this primary antibody is conjugated to a fluorophore, and (ii) indirect IF uses an unconjugated primary and a fluorophore-conjugated secondary antibody, specific to the primary antibody, to allow detection.
A. Preparation
Sample Preparation
- Incubate cover glass in 50% H2SO4 for 1 hr, and then rinse with sterile H20.
- Incubate coverslips with Poly-L-lysine for 1hr, at room temperature.
- Rinse cover glass well with sterile H2O (3 times, 1 hr each).
- Allow to dry, then sterilise under UV light for 4 hrs.
- Culture cells on cover glass, or prepare cytopsin or smear preparation. Wash briefly with PBS.
Fixation
Methanol Fixation
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Formaldehyde Fixation
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Paraformaldehyde / Glutaraldehydefixation
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EGS (ethyleneglycol-bis-succinimidyl-succinate) Fixation
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Antigen Retrieval (optional)
Some antibodies are more effective when cells are heated in antigen retrieval buffer - check the product information for recommendations for the specific (primary) antibody used.
- Preheat the antigen retrieval buffer by placing it into a cover glass staining jar which is placed into a water bath at 95°C
- Place the cover slips into the cover glass staining jar containing the antigen retrieval buffer
- Heat the coverslips for 10 mins.
- Remove the coverslips and with the side containing the cells facing up, immerse them in PBS.
- Wash cells 3 times In PBS for 5 mins.
Permeabilisation (if needed)
For intracellular targets, it is crucial to permeablise cells. Methanol fixed samples do not require this step.
- Incubate the samples in PBS containing 0.1% Triton X-100 for 10 mins. Note: Percentage of Triton X-100 should be determined for each different protein.
- Wash cells three times in PBS for 5 mins.
B. Procedure
Blocking and Immunostaining
- Singular Staining for ONE target antigen
- Incubate cells with 1% BSA in PBS for 30 mins to block non-specific binding of antibodies.
- Incubate cells with the primary antibody in 1% BSA in PBS overnight at 4°C
- Wash the cells 3 times with PBS for 10 mins.
- Incubate cells with the secondary antibody in 1% BSA in PBS for 1 hr at room temperature, in the dark.
- Wash the cells 3 times with PBS for 10 mins.
- Multicolour Staining for TWO (or more) target antigens
Simultaneous Incubation | Sequential Incubation |
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Counter-staining (optional)
- Incubate cells in 0.1-1 mg/ml Hoechst or DAPI(DNA stain) for 1 min.
- Wash with PBS.
Mounting (optional)
- Mount coverslip in PPD mounting medium (90% glycerol).
- Seal with nail polish to prevent drying and movement.