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Primary cultured astrocytes were incubated with 5 μm each 3a and b for 1 h, washed with PBS, and then click reaction performed with 5 μM Alexa 546 DIBO alkyne for 20 min at RT. After washing, cells were fixed with p-formaldehyde/glutaraldehyde and permeabilized under mild conditions (0.2 % Triton X-100/PBS, 5 min, RT). Immuncytochemistry and co-labeling was performed with anti-ceramide rabbit IgG and Alexa 488 anti-rabbit IgG.
