Mike Barnkob:Protocols/Visualization/Immunofluorescence on cell lines
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.
- 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).
- 10 % poly-L-lysine solution: Dilute Poly-L-lysine 1 in 10 in PBS
- Approximately 200 μL needed per. sample.
- 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.
- 1 ml 4% formaldehyde.
- 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.
- 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.
- 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
- 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.
- 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 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
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.
- "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.
- Protocol for the Preparation and Fluorescent ICC Staining of Non-adherent Cells