Visualizing SGPL1 Cell Membrane Association

General scanning electron microscopy method was described previously in Engel et al., 2016 [28 (link)]. For the visualization of SGPL1 cell membrane association, cells were grown on coverslips to a confluency of 80%. After washing with PBS with Ca²⁺ and Mg²⁺, unspecific binding sites were blocked with 5% goat serum (PAN Biotech, Germany). Primary labeling with SGPL1 antibody (1:100 dilution of SGPL1 in 5% goat-PBS; sc-67368, Santa Cruz, USA) was performed at room temperature for 1 h. Secondary labeling with BBI gold anti-mouse antibody (1:200 dilution in 5% goat-PBS, 0.1% fish skin gelatin (BBI), 0,1% Tween (VWR, Germany); EM GMHL, 15 nm, BBI Solutions Cardiff, UK) was also performed for 1 h. After washing, cells were fixed with 2.5% glutaraldehyde in 0.05 M HEPES buffer. Samples were dehydrated in an ascending ethanol series and critical point dried using CO₂ as an intermedium with the EMITECH 850 critical point dryer (Emitech Ltd. Ashford, UK). Carbon coating was done with the carbon coater SCD500 (Leica, Germany). Images were taken with a field emission scanning electron microscope (Zeiss Merlin VP Compact) using an acceleration voltage of 5 kV for imaging.