Immunogold Labeling Method for Electron Microscopy (for Ultrathin Sections)
(All incubations are at room temperature other than those indicated.)
Fixation, embedding, and sectioning:
- 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.
- 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.
- 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
- 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.
- 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).
- Rinse. Longer incubations with higher dilution of antibody produce more specific labeling.
- 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.
- 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.
- Transfer the grid to a series of 50 µl droplets of distilled water (5 x 2 min) to remove unbound gold conjugate.
- 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.