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Immunocytochemistry and Immunohistochemistry using Fluorescein-labeled Secondary Antibodies

Solutions

  • PBS – sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline
  • Antibody dilution buffer (PBS with 0.1% non-ionic detergent, such as Triton X-100 or Tween-20)
  • fluorescein anti-fading reagent — Make up a 2 mg/ml phenylene diamine solution in PBS (phenylene diamine requires extensive vortexing to put it into solution). Once the phenylene diamine is completely dissolved, add an equal volume of glycerol and mix. This reagent will last about a week at -20C. Discard this reagent when it starts to turn dark brown.

Other Reagents

  • Fluorescein-labeled goat anti-chicken IgY (Aves Labs, Catalog # F-1001)
  • BlokHen® (Aves Labs, Catalog # BH-1001)

Steps

  1. Prepare your tissue sections or cultured cells as you normally would.
  2. Block non-specific binding sites by incubating your sections or cells with BlokHen (1:10 dilution in PBS) for 15 minutes at room temperature.
  3. Wash your sections or cells for 1 min with PBS at room temperature.
  4. Incubate your sections or cells with your chicken primary antibodies (diluted in “antibody dilution buffer”) for at least 1 hour at room temperature. The concentration of your antibody may be anywhere from 1:100 to 1:50000, depending on the titre of the antibody and the concentration of your antigen.
  5. Wash your sections or cells over a 10-minute period at room temperature (with two changes of PBS).
  6. Incubate your sections or cells with fluorescein-labeled goat anti-chicken IgY (1:500 dilution in “antibody dilution buffer) for 1 hour at room temperature. Be sure to keep these slides or culture dishes in subdued light (e.g., in a drawer) to avoid bleaching of the fluorescein dye.
  7. Repeat step #5.
  8. Add a drop of “fluorescence anti-fading reagent” to your sections or cells. Place a coverslip over the section. If you want to reduce messiness, you may also seal the coverslip by painting the edges with nail polish.
  9. Store the slides or culture dishes in the refrigerator (in the dark).

Further Reading on Methods

These methods have been adapted from a variety of sources, including:

  • “Antibodies: A Laboratory Manual” by Harlow and Lane (Cold Spring Harbor Press, 1988);
  • “Current Protocols in Molecular Biology” by Ausubel et al. (Wiley & Sons, 1997); and
  • various Specification Sheets offered by manufacturers.

We highly recommend the manual by Harlow and Lane, which provides general information on antibodies.