Immunofluorescence Protocol

Date Published: July 18, 2023

Principle

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections. Immunofluorescence stained samples are examined under a fluorescence microscope or confocal microscope.

Cell Staining

  1. Fix cells using appropriate method (see fixation protocols)
  2. Block non-specific antibody binding with 3% Bovine Serum Albumin in PBS (w/v) (PBS-BSA) for 30 minutes.
  3. Incubate with primary antibody for 1 hour (primary antibody should be diluted in PBS-BSA).
  4. Wash cells 3 times 5 minutes each in PBS.
  5. Incubate with secondary antibody diluted in PBS-BSA for 1 hour (only if indirect staining is desired, if you are using direct staining, skip this step).
  6. Wash 3 times, 10 minutes each in PBS. Counter stain with DAPI (1:5000 in PBS) if desired.
  7. Mount coverslips on slides with Mowiol where required. Live cell dishes can be left in PBS as long as images are acquired within 24 hours of immunostaining.

Tissue Staining

  1. Fix tissue using appropriate method (see fixation protocols).
  2. Using a hydrophobic wax pen, encircle the tissue section on each slide. This will help prevent excessive spreading of the reagents. Do not allow the sections to dry.
  3. Permeabilize and block tissue sections (permeabilization solution) for 60 minutes at room temperature: using a micropipettor, cover each tissue section (volume will depend on tissue size) with the solution.
  4. Dilute the primary antibody in 1% BSA (Bovine Serum Albumin) and 0.05% Triton® X-100 in PBS. Incubate the samples overnight (in the dark for direct staining) at 4°C.
  5. Wash the sections (3 X 5 min.) in PBS.
  6. Dilute the secondary antibody in 1% BSA and 0.05% Trition X-100 in PBS. Apply the antibody to the samples and allow to incubate for 60 minutes at room temperature in the dark (only if indirect staining is desired, if you are using direct staining, skip this step).
  7. Mount the slides using a mounting medium.
  8. Store in a darkened place to minimize photo bleaching.

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