Mike Barnkob:Protocols/Visualization/Immunofluorescence on cell lines

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Immunofluorescence protocol

This is a protocol for doing IF (immunofluorescence) on adherent and non-adherent cell lines.
This protocol was last updated on 31th of May 2015.

This protocol is not yet complete.

Reagents

  • PBS
  • 100% ethanol
  • Formaldehyde, 40%
  • Trypsin or EDTA
  • Coverslips: round Ø 13 mm for adherent cells (VWR, cat. 631-0141) or 24x60 mm for non-adherent cells (VWR, cat. 631-0150).
  • Microscopy slides (VRW, cat. 631-0909).
  • Antibodies and secondaries
  • Mounting media with DAPI (SouthernBiotech, cat. 0100-20).
  • Poly-L-lysine solution (Sigma, cat. P8920-100ml).

Prepare:

  • 10 % poly-L-lysine solution: Dilute Poly-L-lysine 1 in 10 in PBS
    • Approximately 200 μL needed per. sample.
  • Fixative(s):
    • 1 ml 4% formaldehyde.
      • Dilute 40% formaldehyde 1 in 40
    • Ice-cold 100% acetone, 100% ethanol or 1:1 mix of ethanol/acetone.
      • Keep at -20°C before use.
  • Blocking solution: 1% bovine serum albumin (BSA) in PBS.
    • 1g BSA to 100mL of PBS.
    • BSA is in common reagents fridge.
  • Blocking + permabilization solution: Blocking solution + 0.1% Triton X-100.
    • 50mL of blocking solution + 50μL Triton X-100.
  • Ice cold PBS: keep PBS at 4°C for washing steps.

Controls

  • Negative control: one samples prepared as others, but without antibody staining.
  • Isotype control: one samples prepared as other, but has been stained with an irrelevant antibody of the same isotype which has no specificity to the protein of interest.
  • Secondary control: one samples prepared as other, but stained only with the secondary antibody.

Protocol for adherent cells

Coat cover slips

  • Place cover slips in 24 well plate.
  • Wash cover slips with PBS x 1. Aspirate liquid off by tipping plate in opposite directions, so that pipet-tip does not touch the cover slips.
  • Sterilize cover slips by adding 500 μL of 100% ethanol to each cover slip. Aspirate off and air dry in flow-hood for 15-20 minutes.
  • Incubate cover slips with 200μL 10% poly-L-lysine solution for 30 minutes. Gently rock plate to ensure even distribution. Aspirate off.
  • Wash cover slips with PBS x 2.
  • Plate out cells on coverslip and incubate them at 37°C for 24-48 hours.
    Note: Use 400-500μL of cells with cell media in each well.
    Note: Many protocols call for 70% confluency, but less can do it in my opinion.
  • Aspirate off media and wash in PBS x 2.

Fixation

Two methods:

  • Incubating cells in 100% acetone (chilled to -20°C) for 5 min at room temperature, or:
  • Incubate cells in 4% paraformaldehyde in PBS for 10 min at room temperature.
  • Following either method, wash cells with ice-cold PBS x 3. Aspirate off liquid as before.

Blocking, permeabilization and staining

Primary antibody:

  • Incubate cells in blocking solution either with or without permabilization reagent (Triton X-100) for 20 min at room temperature.
    Note: Acetone fixed samples might not need permeabilization, and so a pure blocking solution can be tried. But really, why not use the permabilization any way?
  • Incubate cells with 1st antibody, diluted in blocking solution, for 1 hour at room temperature in the dark.
    Note: Many protocols recommend incubating over-night at 4°C.
    Note: If staining for intra-cellular antigen, consider using permabilization solution instead.
  • Wash cells with PBS x 3. Aspirate or decant off liquid.

Secondary antibody:

  • Incubate cells with 2nd antibody, diluted in blocking solution, for 1 hour at room temperature in the dark.

Finale wash:

  • Wash cells with PBS x 3. Aspirate off liquid.
  • Wash cell in dH20 x 1.

Counter-stain and mount

  • Mount coverslip with a drop of mounting medium.
    Note: Be careful not to get air bubbles by slowly aspirating, and only adding a small amount on glass slide.
  • Using a pipet, carefully remove cover slip from well plate and place on glass slide at an angle.
  • Seal coverslip with nail polish.
  • Store in dark at -20°C or +4°C.

Protocol for non-adherent cells

Trouble-shooting

For staining cell surface proteins:

  • Consider using 1mM EDTA instead of Trypsin for loosening cells, as Trypsin might strip receptors from the cell surface.
  • By doing the majority of reactions with cold liquids and on ice, internalization of cell-membranes can be lessened.
  • Permabilization with Triton X-100 should be tried with care since it can destroys membranes and thereby also the membrane-associated antigens.


Reference