Immunofluorescence
ReagentVendorCatalog 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:
  • Biosafety Cabinet
  • Sterile, glass, borosilicate pipets (Fisherbrand)
    • 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:
    1. Observe cells under microscope and choose best sample for imaging. Passage or freeze remaining cells
    2. Aspirate media from the cells
    3. Wash with PBS
    4. Pipet enough volume of 4% Paraformaldehyde to cover the surface of the well or plate
    5. Incubate at room temperature for 20 minutes
    6. Aspirate 4% paraformaldehyde from the cells
    7. Pipet enough 5% GS/PBST to be half the volume used for feeding cells in specific well or plate
    8. Incubate room temperature for one hour
    Primary Antibody:
    1. 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)
    2. Aspirate 5% GS/PBST from the cells
    3. APipet primary antibody solution to the cells
    4. Incubate at 4OC overnight or at room temperature for 1 – 2 hours
    Secondary Antibody:
    1. 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.
    2. Aspirate primary antibody solution from the cells
    3. Wash with 0.3% PBST for 5 minutes, three times
    4. Pipet secondary antibody solution to the cells
    5. Incubate at room temperature for 1 – 2 hours in the dark
    6. Prepare DAPI solution; 1:10,000 diluted in PBS. Keep solution in the dark at 4OC until use
    7. Aspirate and Wash with 0.3% PBST for 5 minutes, twice
    8. Pipet DAPI solution onto cells
    9. Incubate room temperature for 5 minutes
    10. Aspirate and wash with PBS
    11. View under fluorescent microscope