Immuno-SEM

Thanks for interest in the manuscript. The immuno-SEM method we applied used TNM-BF to immunolocalise cholesterol at the cell surface, but this method does also work with other antigens exposed at the surface of cells. We find that the required dilution of primary antibodies is roughly equal to the dilutions used for immunofluorescence.

Cells were fixed using a double strength fixative (8%PFA/PBS) at a 1:1 ratio with culture media (final, 4% PFA/PBS/media). Cells were then quenched with 15mM glycine / PBS for 10mins before being blocked with 1% BSA/PBS for 20mins. 

Coverslips were then incubated with 1 μM TNM-BF for 30 min on ice and in the dark, before being washed 3x with blocking buffer. Coverslips were then incubated with a rabbit polyclonal anti-BODIPY antibody (A-5770, Molecular Probes) - dilution 1/200 for 1 hour. Coverslips were again washed 3x with blocking buffer before being incubated with 10 nm protein-A gold - dilution appropriate to batch (Utrecht). Coverslips were finally washed 3x with blocking buffer and PBS before being re-fixed with 2% PFA, 2.5% glut, 0.1M cacodylated. Coverslips were washed with 0.1M cacodylate buffer and osmicated before being processed and imaged by SEM. A backscatter detector was used to identify gold particles.