Fluoroshield with 4 6 diamidino 2 phenylindole dapi mounting medium

The ACFCs after TSA treatment were washed with sterile PBS and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). After several washes in PBS, cells were blocked in 5% normal goat serum (NGS) in PBST (PBS containing 0.1% Triton X-100) for 30 min. Cells were then incubated overnight at +4 °C in humidified chamber with the following primary antibodies (the same as those for Western blot): against human α1,2-fucosyltransferase (diluted 1:150 in PBST) and human α-galactosidase (diluted 1:200 in PBST). In the next step, cells were washed several times in PBST and treated with goat anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (diluted 1:300 in PBST; ThermoFisher Scientific, Waltham, MA, USA) for 1 h at room temperature. After final washes, cells were mounted in Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (F6057, Sigma-Aldrich). Fluorescently labelled ACFCs were examined as described in Section “4.5. Confocal microscope analyses”.

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).