Immunogold Labeling - Microscopy

Immunogold Labeling Method for Electron Microscopy (for Ultrathin Sections)

(All incubations are at room temperature other than those indicated.)

Fixation, embedding, and sectioning:

  1. Fixing the cells in 2% fresh-made paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at room temperature for 2 hrs.
  2. Rinsing the samples 3 times (10 min each) in 0.1 M phosphate buffer and quenching the aldehyde group with 0.1% fresh-made sodium borohydride for 5 min.
  3. Dehydration of the cells through a graded ethanol series and embedding the samples in LR white based on standard protocol. Ultrathin sections should be collected with nickel grids.

Immunogold labeling and staining

  1. Etching (optional): Place the grid, section face down, on a 25 µl droplet 20% H2O2 for 20 min. Blocking: grid with section face down on a droplet of 5% BSA/Tris buffer saline TBS or BSA/PBS buffer. Incubate for 10-20 min.
  2. Use a platinum wire loop or forceps to transfer the grid to the surface of a 25 µl droplet of primary antibody (in this case, goat anti-ER proteins at 1:100-200) in appropriate buffer (e.g. 1% BSA/TBS). Incubate for 3 hours (or overnight at 4).
  3. Rinse. Longer incubations with higher dilution of antibody produce more specific labeling.
  4. Wash the sections by transferring the grids through 5 (2 min each) droplets (50ul) of 0.1% BSA-TBS. Do not let the sections dry.
  5. Transfer the grid to a 25 µl droplet of anti-goat IgG conjugated with gold (10nm-15nm), diluted based on company's instruction (depending the suppliers) in TBS. Incubate for 1 hour.
  6. Transfer the grid to a series of 50 µl droplets of distilled water (5 x 2 min) to remove unbound gold conjugate.
  7. Stain embedded sections lightly in uranyl acetate and lead citrate (optional). Frozen sections may also be stained in osmium tetroxide vapor. Wash and examine under the electron microscope.