SUT-6 immunofluorescence in C. elegans

For each strain, age synchronized D1A worms were picked and transferred to three successive droplets of M9 then to a drop of 1% PFA on poly-L-lysine-coated slides. The worms were permeabilized by freeze-cracking and fixed by methanol/acetone fixation. Slides with fixed and permeabilized animals were moved to a humidifying chamber, washed once with PBS, then placed in blocking solution (10% goat serum in antibody buffer) for 1 h at room temperature. After blocking, the worms were incubated with SUT-6 6B primary antibody at a dilution of 1:100 and tau monoclonal antibody SP70 (Invitrogen) at 1:1000 in blocking solution at 4°C overnight. Slides were then washed 3× with PBS, 2× with blocking solution, then incubated in AlexaFluor 647F(ab′)2 fragment of goat anti-mouse IgG (H + L) (Jackson Immunological) and AlexaFluor 488F(ab′)2 fragment of goat anti-rabbit IgG (H + L) (Jackson Immunological) both at 1:1000 in blocking solution at room temperature for 1 h. Slides were then washed 3× with PBS, incubated with 300 nm DAPI for 5 min at room temperature, washed 2× more with PBS, then mounted with ProLong Gold (Thermo Fisher Scientific). Animals were imaged with a DeltaVision Elite fluorescent microscope (Applied Precision). Images shown in Figure 4C were threshold-adjusted consistently across the entire image in Photoshop to reduce background signal and allow for better visualization of nuclei in print.