Immunocytochemistry Protocol for Protein Visualization

Immunocytochemistry was performed using previously described standard techniques [11 (link),18 (link)]. Briefly, cells were fixed with 4% paraformaldehyde solution, permeabilized with a nonionic surfactant, and blocked using bovine serum albumin or fetal bovine serum for gels. Cells were then incubated sequentially in primary and fluorescent secondary antibody solutions to allow visualization of targeted proteins by fluorescence microscopy (full details, antibody sources, and concentrations included in S1 Supporting information). Secondary antibody-only controls were performed by omitting the primary antibody during incubation (S1 Fig). For positive controls for immunofluorescence staining of ONPs refer to previous work [8 (link)]. Fluorescence imaging was performed on a Zeiss UV-LSM 510 META or a Nikon A1/C2 confocal microscope. Sequential scanning of channels was performed to prevent false-positive co-localization. ImageJ 1.51e 5 [33 (link)] was used to quantify images.