Mike Barnkob:Protocols/Visualization/Immunofluorescence on cell lines: Difference between revisions

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* PBS
* PBS
* Formaldehyde, 16%
* Formaldehyde, 16%
* Trypsin or PBS


'''Prepare:'''
'''Prepare:'''
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* Blocking solution: 1% bovine serum albumin (BSA) in PBS.
* Blocking solution: 1% bovine serum albumin (BSA) in PBS.
** 1g BSA to 100ml of PBS.
** 1g BSA to 100ml of PBS.
** BSA is in common reagens fridge.
** BSA is in common reagents fridge.
* Blocking + permabilization solution: Blocking solution + 0.1% Triton X-100.


===Setup and controls===
===Setup and controls===
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===Protocol for adherent cells===
===Protocol for adherent cells===
 
====Coat 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.
 
====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.
 


====Fixation====
====Fixation====
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* [http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4440.full?sid=0514f1a5-95f7-459b-8ec2-ecf5e30a436e "Fluorescent Staining of Cell Surface Antigens on Viable Cells Using Indirect Immunofluorescence", Rodgers L, 2006, CSH Protocols]
* [http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4440.full?sid=0514f1a5-95f7-459b-8ec2-ecf5e30a436e "Fluorescent Staining of Cell Surface Antigens on Viable Cells Using Indirect Immunofluorescence", Rodgers L, 2006, CSH Protocols]
* [http://bitesizebio.com/13460/how-to-fix-adherent-cells-for-microscopy-and-imaging/ "How To Fix Adherent Cells For Microscopy And Imaging", Redig J, 2013]
* [http://bitesizebio.com/13460/how-to-fix-adherent-cells-for-microscopy-and-imaging/ "How To Fix Adherent Cells For Microscopy And Imaging", Redig J, 2013]
* [http://www.biomedcentral.com/1756-0500/6/317 "Immunofluorescence labeling of cell surface antigens in Dictyostelium", Vernay A and Cosson P, BMC Research Note, 2013]
* [http://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol Abcam's immunofluorescence (IF) protocol]

Revision as of 03:08, 27 May 2015

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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).
  • PBS
  • Formaldehyde, 16%
  • Trypsin or PBS

Prepare:

  • Fixative: 1 ml 4% formaldehyde.
    • 250 ul 16% formaldehyde in 750 ul PBS.
  • 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.

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.

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.


Fixation

Permeabilization

Block

Incubate with antibodies

Counter-stain

Mount

Protocol for non-adherent cells

Reference

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