Immunolabelling techniques

Immunofluorescence

Overview of antibody immunolabelling method

  1. Block non-specific binding sites of prepared cells or a section of plant material adhered to a glass slide by incubation with 3% (w/v) milk protein in phosphate-buffered saline (MP/PBS) for at least 30 min.
  2. Incubate with rat monoclonal antibody diluted in MP/PBS for at least 1 hr at RT or overnight at 4oC. A five-fold dilution of a hybridoma supernatant is a good starting point for the primary antibody.
  3. Wash with three changes of PBS with at least 5 min for each change.
  4. Incubate with a secondary antibody diluted in the region of 100-fold in MP/PBS for 1 hr at RT.  We use anti-rat-IgG (whole molecule) linked to FITC (Sigma)
  5. Wash with three changes of PBS with at least 5 min for each change.

Mount slides using an antifade reagent and examine. We use Citifluor glycerol-based antifade for sections.

Immunogold

Overview of antibody immunolabelling method

  1. Block to prevent non-specific binding by floating the EM grid section side down on a droplet (at least 20 ml) of 3% (w/v) bovine serum albumin in phosphate-buffered saline (BSA/PBS) on Parafilm for 30 min.
  2. Transfer grid to a droplet of primary antibody diluted in BSA/PBS. Monoclonal antibodies should be diluted between 5-fold and 100-fold.
  3. Wash grids by incubation in a minimum of three changes of PBS.
  4. Transfer grids to secondary antibody diluted 1 in 20 with BSA/PBS. We use anti-rat IgG coupled to 10 nm gold (Sigma).
  5. Wash as in step 3 and then extensively in distilled water.
  6. Allow the grid to dry and then examine in an electron microscope.