Correlative Light-Electron Microscopy of Cellular Organelles

Using a modified method (77 (link), 78 (link)), we washed cells 3 times for 5 minutes with PBS with 0.02 M glycine before incubating for 1 hour with anti-LAMP1 (Pharmingen, 1:50). We then washed cells 3 times for 10 minutes with PBS with 0.02 M glycine. The grids were incubated for 20 minutes with a goat anti–mouse Alexa Fluor 488 secondary antibody (Life technologies, Thermo Fisher Scientific, 1:500) and for 10 minutes with 1 mg/mL Nile red (MilliporeSigma, 1:25) and Hoechst 33342 (Thermo Fisher Scientific, 1:50). Finally, the grids were washed 5 times with PBS. The grids were mounted in between a glass slide and a coverslip in a droplet of VECTASHIELD and then imaged (Leica DM6, 100× oil objective). After wide-field imaging, the coverslip was removed, and the VECTASHIELD was washed from the grid with milliQ water at 37°C. Thereafter, the grids were contrasted using uranyl acetate in 2% methylcellulose for 5 minutes, blotted, and imaged (Fei Tecnai 120kV) with correlation (ICY Software) and CLEM (Huygens) deconvolution software.