Immunofluorescence

1. Sample Preparation

    a) Incubate cover glass in 50% H2SO4for 1 hour using a porcelain rack.
    b) Rinse cover glass with sterile H2O
    c) Incubate coverslips with Poly-L-lysine for 1hr at room temperature.
    d) Rinse cover glass well with sterile H2O(three times, 1hr each).
    e) Allow to dry and sterilise under UV light for at least 4hrs.
    f) Culture cells on cover glass or prepare cytospin or smear preparation.

OR

    a) Detach cell from the plastic surface by incubating them in trypsin solution.
    b) Resuspend detached cells in culture medium and transfer them to culture dishes with the cover glasses.
    c) Culture cells up to semi-confluency.
    d) Drain off culture medium and rinse cover slips with PBS.

2. Fixation

Methanol Fixation

    a) Rinse the cover glass with PBS.
    b) Fix cells by incubating the cells in -20oC 100% methanol for 10 minutes.
    c) Then wash cells three 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) Then wash cells three times with PBS containing 100 mM glycine.

OR

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 three 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) Rinse three times in PBS.

3. Antigen retrieval (optional)

  • a) Preheat the antigen retrieval buffer by placing it into a cover glass staining jar which is placed into a water bath at 95oC
  • b) Place the cover slips into the cover glass staining jar containing the antigen retrieval buffer.
  • c) Heat the coverslips for 10 min
  • d) Remove the coverslips and with the side containing the cells facing up, immerse them in PBS.
  • e) Wash cells three times In PBS for 5 min

4. Permeabilisation (if needed)

  • a) Incubate the samples in PBS containing 0.1%Triton X-100 for 10 min.
  • b) Percentage of Triton X-100 should be determined for each different protein.
  • c) Wash cells three times in PBS for 5 min

5. Blocking and immunostaining

Singular staining

    a) Incubate cells with 1% BSA in PBS for 30 min to block unspecific binding of antibodies.
    b) Incubate cells with the primary antibody in 1% BSA in PBS overnight at 4o.
    c) Wash the cells three times with PBS for 10 min.
    d) Incubate cells with the secondary antibody in 1%BSA in PBS for 60 min at room temperature, in the dark.
    e) Wash the cells three times with PBS for 10 min.

OR

Simultaneous incubation

  • a) Incubate cells with blocking solution (1% BSA in PBS) for 30 min.
  • b) Incubate cells with both of the primary antibodies in 1% BSA in PBS overnight at 4oC.
  • c) Wash cells three times in PBS for 5 min each.
  • d) Incubate cells with both of the secondary antibodies in 1% BSA in PBS for 60 min at room temperature in the dark.
  • e) Wash cells three times in PBS for 5 min each.

Sequential incubation

    a) First serum blocking-Incubate cells in serum that is the same species as the secondary antibody for 30 min at room temperature.
    b) Incubate cells with the first primary anti body in 1% BSA in PBS overnight at 4oC.
    c) Wash cells three times in PBS for 5 min each.
    d) Incubate cells with the first secondary anti body in 1% BSA in PBS for 60 min at room temperature in the dark.
    e) Wash cells three times in PBS for 5 min.
    f) Second serum blocking-Incubate cells in serum that is the same species as the secondary antibody for 30 min at room temperature in the dark.
    g) Incubate cells with the second primary antibody in 1% BSA in PBS over night at 4oC.
    h) Wash cells three times in PBS for 5 min.
    i) Incubate cells with the second secondary antibody in 1% BSA for 60 min at room temperature in the dark.

6. Counter staining (optional)

  • a) Incubate cells in 0.1-1 mg/ml Hoechst or DAPI(DNA stain) for 1 min.
  • b) Rinse with PBS.

7. Mounting (optional)

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