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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

  1. Incubate cover glass in 50% H2SO4 for 1 hr, and then rinse with sterile H20.
  2. Incubate coverslips with Poly-L-lysine for 1hr, at room temperature.
  3. Rinse cover glass well with sterile H2O (3 times, 1 hr each).
  4. Allow to dry, then sterilise under UV light for 4 hrs.
  5. Culture cells on cover glass, or prepare cytopsin or smear preparation. Wash briefly with PBS.

Fixation

Methanol Fixation

  • a) Rinse the cover glass with PBS.
  • b) Fix cells by incubating at -20oC in 100% methanol for 10 minutes.
  • c) Wash cells 3 times with ice-cold PBS.

Formaldehyde Fixation

  • a) Rinse cells with PBS at room temperature.
  • b) Fix in 3-4% paraformaldehyde in PBS for 15 min at room temperature.
  • c) Wash cells 3 times with PBS containing 100 mM glycine.

Paraformaldehyde / Glutaraldehydefixation

    a) Rinse cells with PBS at room temperature.
  • b) Fix cells with 3% paraformaldehyde, 0.02% glutaraldehyde in PBS for
    15 min at room temperature.
  • c) Wash cells 3 times in PBS containing 100 mM glycine.

EGS (ethyleneglycol-bis-succinimidyl-succinate) Fixation

  • a) Rinse cells with pre-warmed PBS.
  • b) Dilute EGS stock in PBS to 10 mM.
  • c) Drain excess PBS from cells and transfer into EGS in under one min.
  • d) Incubate at 37oC for 10 min.
  • e) Wash cells 3 times in PBS.

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.

  1. Preheat the antigen retrieval buffer by placing it into a cover glass staining jar which is placed into a water bath at 95°C
  2. Place the cover slips into the cover glass staining jar containing the antigen retrieval buffer
  3. Heat the coverslips for 10 mins.
  4. Remove the coverslips and with the side containing the cells facing up, immerse them in PBS.
  5. 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.

  1. 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.
  2. Wash cells three times in PBS for 5 mins.


B. Procedure

Blocking and Immunostaining

  • Singular Staining for ONE target antigen
  1. Incubate cells with 1% BSA in PBS for 30 mins to block non-specific binding of antibodies.
  2. Incubate cells with the primary antibody in 1% BSA in PBS overnight at 4°C
  3. Wash the cells 3 times with PBS for 10 mins.
  4. Incubate cells with the secondary antibody in 1% BSA in PBS for 1 hr at room temperature, in the dark.
  5. Wash the cells 3 times with PBS for 10 mins.
  • Multicolour Staining for TWO (or more) target antigens
Simultaneous Incubation Sequential Incubation
  1. Incubate cells with blocking solution (1% BSA in PBS) for 30 mins.
  2. Incubate cells with both of the primary antibodies in 1% BSA in PBS overnight at 4°C.
  3. Wash cells 3 times in PBS, for 5 mins each.
  4. Incubate cells with both of the secondary antibodies in 1% BSA in PBS for 1 hr at room temperature in the dark.
  5. Wash cells 3 times in PBS for 5 mins each.
  1. First serum blocking: Incubate cells in serum (same species as the secondary antibody) for 30 mins at room temperature.
  2. Incubate with primary antibody in 1% BSA in PBS overnight at 4°C.
  3. Wash 3 times (for 5 mins) with PBS.
  4. Incubate with secondary antibody in 1% BSA in PBS for 1 hr (room temperature) in the dark.
  5. Wash 3 times (for 5 mins) with PBS.
  6. Second Serum Blocking: Repeat Steps 1-5 for the rest of the primary/secondary antibodies against the different target antigens.


Counter-staining (optional)

  1. Incubate cells in 0.1-1 mg/ml Hoechst or DAPI(DNA stain) for 1 min.
  2. Wash with PBS.

Mounting (optional)

  1. Mount coverslip in PPD mounting medium (90% glycerol).
  2. Seal with nail polish to prevent drying and movement.