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Immunofluorescence (IF) - Protocol

Immunofluorescence is an immunostaining technique that visualizes antigens in samples using fluorescent microscopy. Samples are fixed carefully with organic solvents and chemical crosslinkers to maintain their cellular integrity as well as sub-cellular architecture. They are then permeabilized to allow immunostaining and then blocked to minimize non-specific binding. Samples are then immunostained with antibodies and via fluorescent reaction targets are visualized in color.

Materials:

  • 10X PBS: To prepare 1L, add 80g NaCl, 2g KCl, 2g KH2PO4 and 28.5g NaHPO4 to 1L water for injection. Adjust pH to 7.4.
  • 4% Polyoxymethylene: To prepare 100mL, add 4g polyoxymethylene to 100mL 1×PBS. Adjust pH to 7.4.
  • 1×PBS/0.2% Triton X-100(PBS/Triton): To prepare 500mL, add 1mL Triton X-100 to 500mL 1× PBS.
  • 1×PBS/3% BSA(PBS/BSA): To prepare 100mL, add 3g BSA to 100mL 1× PBS.

Protocol:

  • Preparation Fixation permeabilization
  • 1. Rinse cells briefly in PBS.
  • 2. Aspirate PBS, cover cells to a depth of 2-3mm about 200ul with 4% polyoxymethylene.
  • 3. Allow cells to fix for 15 minutes at room temperature.
  • 4. Aspirate fixative, rinse three times in PBS for 5 minutes each.
  • 5. Aspirate PBS, cover cells to a depth of 2-3mm about 200ul with PBS/Triton for 5 minutes at room temperature.
  • 6. Aspirate permeability agent rinse three times in PBS for 5 minutes each.

  • Immunostaining
  • 1. Gently add 200ul of primary antibody diluted in PBS/BSA to the 24 well plates each well.
  • 2. Incubate 60 minutes at 37℃ or overnight at 4℃.
  • 3. Aspirate diluted primary antibody, then rinse three times in PBS for 5 minutes each.
  • 4. Incubate in fluorochrome-conjugate secondary antibody diluted in PBS/BSA to the 24 well plates 100ul each well for 30 minutes at room temperature in dark.
  • 5. Aspirate diluted fluorochrome-conjugate secondary antibody, then rinse three times in PBS for 5 minutes each.
  • 6. Test under fluorescenct microscope.