Immunofluorescence Protocol Started 3-21-12

2015-01-13

azim58 - Immunofluorescence Protocol Started 3-21-12

Immunofluorescence Protocol Started 3-21-12

Materials

o Sterile glass cover slip
- (what brand? cat no?)
- found in tissue culture room?

Place sterile glass cover slip in the bottom of well in a 6-well

culture plate

Plate about 2e5 cells in appropriate media on a 6 well plate in a 2

mL volume. Note that it is best to add 1 mL of media and then add the 1
mL of cells to this down along the side of the well

Grow overnight at 37 C 5% CO2
wash cells with 1X PBS
Fix cells for imaging

*Wash cells 2x with PBS. Add 2ml ice cold 100% MeOH, 5min at -20º,
aspirate MeOHLet air dry 20-30min.

Wash three times with PBS.
*Store in 0.02% (w/v) sodium azide in PBS at 4 degrees for no more than
3 days

Permeabilize Cells:
Incubate the cells in 2ml 0.1% TritonX-100 in PBS for 15min. at room
temperature. Rinse cells 3 times with PBS. (50ul of TritonX 100 into
50ml of 1xPBS)

Block cells:
Incubate specimens with 2ml 10% horse serum in PBS (5ml horse serum in
45ml 1XPBS) for 20 minutes to suppress non-specific binding of IgG.
Blocking serum ideally should be derived from the same species in which
the secondary antibody is raised. Wash once with 1XPBS.

Adding Primary:
Add 1.5ml of the primary in PBS with 1.5% horse serum at a 1:100
dilution to corresponding wells.
Incubate room temperature for 1hr. Rinse 3 times with 3ml of PBS.

Adding Secondary:
Add secondary antibody with dye at 1:2000 to corresponding wells in
1.5% horse serum in PBS.
Incubate room temperature in the dark for 1 hour.

Wash 3 times with PBS.

Adding Rhodamine Phalloidin for actin staining: (optional)
Add 200ul of 100nM rhodamine phalloidin (dilute 3.5ul of 14uM into
500ul PBS). Incubate in the dark at room temperature for 30min. (in a
humid chamber). This dye will be visible in the 555 range.
Wash 3 times with PBS

DAPI Staining for DNA staining DNA**
Warm DAPI from -20 degrees to room temperature. Place one drop of DAPI
on a glass slide. Place glass cover slip one slide with cells touching
the DAPI. Be careful to avoid bubbles between in cover slip and slide.
Fix cover slip to slide with nail polish along the edges. Store in the
dark. DAPI will be visible at 480 nm.

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Other Information

Original Protocols from Kari

o Immunofluor for adherent 4T1 cells protocol.doc
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1338\Immunofluor for adherent 4T1 cells protocol.doc"
o Immunofluorescence Protocol for Suspension Cells.pdf
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1338\Immunofluorescence Protocol for Suspension Cells.pdf"