2.9. High-Performance Thin-Layer Chromatography (HPTLC) and Immunostaining

The total gangliosides of an equivalent of 2 × 10⁶ cells or 0.2 µg of the indicated ganglioside standards were spotted on Nano-DURASIL-20 (0.2 mm silica gel 60) HPTLC plates (Macherey–Nagel) and then chromatographed in chloroform/methanol/H₂O (50:40:10, v/v/v) containing 0.05% calcium chloride. HPTLC plates were dried and chromatographed twice in 0.5% poly(isobutyl methacrylate) (Sigma-Aldrich, Taufkirchen, Germany) in hexane, which was prepared from a 25% stock solution in chloroform (w/v). The plates were dried and incubated overnight at 37 °C in PBS. After blocking with 2% BSA (w/v) in PBS for 1 h at room temperature, the plates were incubated with the following primary antibodies diluted in PBS: mouse IgG3 anti-9-OAcGD2 mAb 8B6 (10 µg/mL; OGD2 Pharma), mouse IgM anti-9-OAcGD3 mAb M-T6004 (1:40; Thermo Scientific, MA1-34707), mouse IgG2a anti-GD2 mAb ME361 (15 µg/mL; Kerafast, Boston, MA, USA; EWI023), or mouse IgG3 anti-GD3 mAb R24 (10 µg/mL; purified by protein A affinity chromatography from cell culture supernatant of R24 hybridoma cells ATCC HB-8445). HPTLC plates were washed three times with PBS and then incubated for 1 h at room temperature with goat anti-mouse IgM IRDye 800CW-conjugate (1:20,000; 926-32280; LI-COR Biosciences, Lincoln, NE, USA) or goat anti-mouse IgG IRDye 800CW-conjugate (1:10,000; 926-32210; LI-COR Biosciences, Lincoln, NE, USA). The HPTLC plates were washed with PBS and bound antibodies were detected by infrared imaging using an Odyssey Imaging System (LI-COR Biosciences).