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 27th of May 2015.

This protocol is not yet complete.

Reagents

• 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
• PBS
• Formaldehyde, 16%
• Trypsin or EDTA

Prepare:

• Fixative(s):
  • 1 ml 4% formaldehyde.
    • 250 ul 16% formaldehyde in 750 ul PBS.
  • Ice-cold 100% methanol, 100% ethanol or 1:1 mix of ethanol/methanol.
    • 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.

Setup and 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

General notes

For staining cell surface proteins:

• Consider using 1mM EDTA instead of Trypsin for loosening cells, as Trypsin might strip receptors from the cell surface.
• The addition of sodium azide can be considered, to stop internalization of receptors; but this is also toxic to cells.
• Another approach for stopping internalization of receptors is to keep the cells on ice at all times and do spins at 4°C.
• Permabilization with Triton X-100 should be tried with care since it can destroys membranes and thereby also the membrane-associated antigens.

Coat glass slide

• Place cover slips in 24 well plate.
• Rinse cover slips with PBS and remove. Repeat 2 times. Wait 5 minutes between rinses.
• (Sterilize cover slips by leaving under tissue culture hood UV light for 15 min.)
• Plate out cells on coverslip and incubate them at 37°C for 24 hours.
  Note: Use 400-500μL of cells with cell media in each well.

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 or decant off liquid.

Blocking, permeabilization and staining

Acetone fixed samples might not need permeabilization, and so a pure blocking solution can be tried.

Primary antibody:

• Incubate cells in blocking solution either with or without permabilization reagent (Triton X-100) for 20 min at room temperature.
• 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.
• Wash cells with PBS x 3. Aspirate or decant off liquid.

Counter-stain and mount

• Incubate cells on 0.1-1 μg/ml Hoechst or DAPI for 1 min.
• Wash cells with PBS x 1. Aspirate or decand off liquid.
• Mount coverslip with a drop of mounting medium.
• Seal coverslip with nail polish.
• Store in dark at -20°C or +4°C.

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.