Immunofluorescence with biotinylated antibodies (Stukenberg lab)

Protocol submitted by VGP from Stukenberg lab protocols [1]

link to protocol page [2]

Immunofluorescence with Biotinylated Antibodies

1. Fix cells onto coverslips as normal, and wash appropriately.

2. Add to your normal first block (20% serum, or 3%BSA, etc., in your buffer of interest; I use PBST or TBST depending on the antibody), approximately 10ul/100ul of Avidin blocking solution (Vector Laboratories, catalog # SP-2001) Block as usual, ~30 minutes at room temperature.

3. Incubate your coverslips in your 1st rabbit primary antibody as usual, (5% serum in your chosen buffer, etc.) at room temperature for 1hour.

4. Wash the coverslips twice for 5 minutes each in PBST or your buffer of choice. I rock my coverslips gently in 6 or 12 well dishes on a rotator.

5. Add 10uL/100uL of Biotin blocking solution to the 1st anti-rabbit 2? antibody, and label the coverslips as usual (5% serum in your chosen buffer), ~45 minutes at room temperature in the dark to protect the fluorophore.

6. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer. Keep the coverslips covered (I use some foil) to protect your fluorophore.

7. This is the critical step: Block the coverslips in rabbit serum or IgG. This will block any leftover IgG from the first anti-rabbit secondary antibody that might be recognized by the biotinylated rabbit antibody that you will use in the next step. If you forget this step, your results will be skewed significantly by the high background staining resulting from the biotinylated rabbit antibody binding to your anti-rabbit secondary used in the previous step.

8. Probe the coverslips with your biotinylated 2nd primary rabbit antibody (5% serum in buffer), 1 hour at room temperature in the dark.

9. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer. Keep the coverslips covered to protect your fluorophore.

10. Incubate the coverslips with Avidin-Cy3 or Avidin-FITC as your 2nd rabbit antibody secondary, for ~45 minutes at room temperature in the dark. (I generally use Avidin-Cy3 (Sigma E-4142) at 1:5,000).

11. Wash coverslips as before, then wash coverslips 1X with ddH2O.

13. Stain coverslips with DAPI or Hoescht 33342.

14. Mount coverslips with mounting media onto slides and seal them.