Immunofluorescent Imaging of Golgi B3Galt6

Immunofluorescent imaging was carried out according to standard methods as described in [15 (link)]. Cells were fixed with 4% paraformaldehyde in PBS. For visualising intracellular markers, cells were permeabilised wit 0.25% Nonidet P-40 alternative in NETGEL buffer (150 mM NaCl, 5mM EDTA, 50 mM Tris-Cl, pH 7.4, 0.05% Nonidet P-40 alternative, 0.25% gelatin and 0.02% sodium azide). Antibodies used in this study were: B3Galt6 (H00126792-B01P, Biotechne), Giantin (ab80864, abcam), Laminin α4 (AF7340, Biotechne), Δ-HS (F69-3G10, AMS Biotechnology), perlecan (7B5, ThermoFisher Scientific), TRITC-conjugated phalloidin (FAK100, Merck), Alexa Fluor 594 goat anti-rabbit (A11012), Alexa Fluor 488 donkey anti-mouse (A21202) and Alexa Fluor 488 donkey anti-sheep (A11015) from Invitrogen/ThermoFisher Scientific. For B3Galt6 intensity in Golgi apparatus, the region of interest per cell was defined by Giantin-positive staining using ImageJ selection tools. The integrated density of B3Galt6 fluorescence of selected regions and background reading were then measured and the difference between the two numbers were corrected total cell fluorescence.