Immunofluorescence Staining of DGDG

Immunofluorescence was performed as previously described (113 (link)). Briefly, cells were fixed in 4% paraformaldehyde for 30 min, permeabilized for 10 min on ice with 0.1% Igepal CA-630 (Sigma-Aldrich) in PHEM buffer (pH 6.9) [60 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂], and background was masked with 3% bovine serum albumin (BSA) in PHEM buffer. DGDG was detected using a polyclonal rabbit anti-DGDG antibody (1:25), a kind gift from Cyrille Y. Botté (University of Grenoble I, Grenoble, France), followed by incubation with a secondary Cy3-labeled polyclonal goat anti-rabbit antibody (AP132C, 1:800, Merck Millipore, Burlington, MA, USA). Cells were mounted on slides using Fluoroshield with 4′,6′-diamidino-2-phenylindole (DAPI) mounting medium (Sigma-Aldrich) and observed with an Olympus BX53 microscope (Olympus, Tokyo, Japan).