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.

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

• "Fluorescent Staining of Cell Surface Antigens on Viable Cells Using Indirect Immunofluorescence", Rodgers L, CSH Protocols, 2006.
• "How To Fix Adherent Cells For Microscopy And Imaging", Redig J, Bitesize Bio, 2013.
• "How To Fix Suspension Cells For Microscopy And Imaging", Redig J, Bitesize Bio, 2013.
• "Immunofluorescence labeling of cell surface antigens in Dictyostelium", Vernay A and Cosson P, BMC Research Note, 2013.
• Abcam's immunofluorescence (IF) protocol.
• Cell Signaling Immunofluorescence Protocol.