Immunofluorescence
Reagent | Vendor | Catalog Number |
---|---|---|
Falcon™ Polystyrene Microplates | Fisher Scientific | Various |
4% Paraformaldehyde (formaldehyde) aqueous solution | Electron Microscopy Sciences | 157-4 |
Goat Serum, New Zealand origin | Thermo Fisher / Life Technologies | 16210064 |
Triton™ X-100 | Sigma Aldrich | T8787 |
Corning™ Cell Culture PBS (1X) | Thermo Fisher / Life Technologies | MT21040CV |
Primary Antibodies | Various | Various |
Secondary Antibodies | Life Technologies | Various |
DAPI | Sigma Aldrich | D9542 |
Thermo Scientific™ ART™ SoftFit-L™ Filtered Pipette Tips | Thermo Scientific | Various |
Required Equipment / Consumables:
Prepare:
0.3% PBST
- Triton X-100 – 150 uL
- PBS – 50 mL
5% Goat Serum/PBST:
- Goat Serum – 500 uL
- 0.3% PBST – 9.5 mL
Blocking Cells:
- Observe cells under microscope and choose best sample for imaging. Passage or freeze remaining cells
- Aspirate media from the cells
- Wash with PBS
- Pipet enough volume of 4% Paraformaldehyde to cover the surface of the well or plate
- Incubate at room temperature for 20 minutes
- Aspirate 4% paraformaldehyde from the cells
- Pipet enough 5% GS/PBST to be half the volume used for feeding cells in specific well or plate
- Incubate room temperature for one hour
Primary Antibody:
- During wait time, prepare your primary antibody solution as suggested by the vendors’ recommendation. Dilute primary antibody with 5% GS/PBST, enough volume to cover the surface of the well or plate (to preserve volume of antibody)
- Aspirate 5% GS/PBST from the cells
- APipet primary antibody solution to the cells
- Incubate at 4OC overnight or at room temperature for 1 – 2 hours
Secondary Antibody:
- Prepare your secondary antibody solution as suggested by vendors’ recommendation. Dilute secondary antibody with 5%GS/PBST, enough to cover the surface of the well or plate (to preserve volume of antibody). Keep solution in the dark at 4OC until use.
- Aspirate primary antibody solution from the cells
- Wash with 0.3% PBST for 5 minutes, three times
- Pipet secondary antibody solution to the cells
- Incubate at room temperature for 1 – 2 hours in the dark
- Prepare DAPI solution; 1:10,000 diluted in PBS. Keep solution in the dark at 4OC until use
- Aspirate and Wash with 0.3% PBST for 5 minutes, twice
- Pipet DAPI solution onto cells
- Incubate room temperature for 5 minutes
- Aspirate and wash with PBS
- View under fluorescent microscope