Immunofluorescence Staining Antibodies Protocol

We used a mouse monoclonal antibody to β-Actin (clone AC-74; Sigma-Aldrich), a mouse monoclonal antibody to Giantin (clone G1/133; Enzo Life Sciences), a rabbit polyclonal antibody to GOLPH3 (Abcam, cat # ab98023), and a sheep polyclonal antibody to TGN46 (AbD Serotec, cat # AHP500G). We also used a homemade, mouse polyclonal antibody to human GOLPH3 that was described before [34 (link)], and a rabbit serum to GFP that was also described elsewhere [35 ]. HRP–conjugated secondary antibodies were from Jackson ImmunoResearch. The following fluorochrome-conjugated antibodies were from ThermoFisher: Alexa Fluor-594–conjugated donkey anti-rabbit IgG, Alexa Fluor-647–conjugated donkey anti-mouse IgG, Alexa Fluor-350–conjugated donkey anti-sheep IgG and Alexa Fluor-647–conjugated donkey anti-sheep IgG. Primary antibodies were used at a dilution 1/200 to 1/2000. HRP–or Alexa Fluor–conjugated secondary antibodies were used at dilutions 1/1000 to 1/20000, depending on their reactivity. The fluorescent nuclear stain 4’,6-diamidino-2-phenylindole (DAPI) was also from ThermoFisher.