Mike Barnkob:Protocols/Visualization/Immunofluorescence on cell lines: Difference between revisions
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==Immunofluorescence protocol== | ==Immunofluorescence protocol== | ||
This is a protocol for doing IF (immunofluorescence) on adherent and non-adherent cell lines. | This is a protocol for doing IF (immunofluorescence) on adherent and non-adherent cell lines.<br /> | ||
This protocol was last updated on 31th of May 2015. | |||
This protocol was last updated on | |||
'''This protocol is not yet complete.''' | '''This protocol is not yet complete.''' | ||
Line 11: | Line 10: | ||
===Reagents=== | ===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). | * 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). | * Microscopy slides (VRW, cat. 631-0909). | ||
* Antibodies and secondaries | * Antibodies and secondaries | ||
* | * Mounting media | ||
'''Prepare:''' | '''Prepare:''' | ||
* Fixative(s): | * Fixative(s): | ||
** 1 ml 4% formaldehyde. | ** 1 ml 4% formaldehyde. | ||
*** | *** Dilute 40% formaldehyde 1 in 40 | ||
** Ice-cold 100% | ** Ice-cold 100% acetone, 100% ethanol or 1:1 mix of ethanol/acetone. | ||
*** Keep at -20°C before use. | *** Keep at -20°C before use. | ||
* Blocking solution: 1% bovine serum albumin (BSA) in PBS. | * Blocking solution: 1% bovine serum albumin (BSA) in PBS. | ||
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* Ice cold PBS: keep PBS at 4°C for washing steps. | * Ice cold PBS: keep PBS at 4°C for washing steps. | ||
=== | ===Controls=== | ||
* Negative control: one samples prepared as others, but without antibody staining. | * Negative control: one samples prepared as others, but without antibody staining. | ||
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===Protocol for adherent cells=== | ===Protocol for adherent cells=== | ||
====Coat cover slips==== | |||
====Coat | |||
* Place cover slips in 24 well plate. | * 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. | ||
* Plate out cells on coverslip and incubate them at 37°C for 24 hours. <br />'''Note:''' Use 400-500μL of cells with cell media in each well. | * Incubate cover slips with poly-L-lysine for 15 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. <br />'''Note:''' Use 400-500μL of cells with cell media in each well. <br /> '''Note:''' Many protocols call for 70% confluency, but less can do it in my opinion. | |||
====Fixation==== | ====Fixation==== | ||
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* Incubating cells in 100% acetone (chilled to -20°C) for 5 min at room temperature, or: | * 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. | * 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 | * Following either method, wash cells with ice-cold PBS x 3. Aspirate off liquid as before. | ||
====Blocking, permeabilization and staining==== | ====Blocking, permeabilization and staining==== | ||
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===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. | |||
Revision as of 08:11, 31 May 2015
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
Prepare:
- 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.
- 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.
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 poly-L-lysine for 15 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.
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
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.
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
- "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.