A 21072

Larval and adult brains were dissected and fixed in 4% formaldehyde in PBS with 0.2% Triton X-100 for 20 min at room temperature. For larval brains, samples were incubated with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 2 h. For adult brains, samples were incubated with primary antibodies at 4°C for 2 nights and secondary antibodies at room temperature for 2 h. The following primary antibodies were used at the indicated dilutions: rat anti-Elav [Developmental Studies Hybridoma Bank (DSHB), 7E8A10; 1:50], mouse anti-Prospero (DSHB, MR1A; 1:10), mouse nc-82 (DSHB; 1:20), rabbit anti-phosphohistone H3 (Cell Signaling, 9701S; 1:200), chicken anti-GFP (Abcam, 13970; 1:2000), rabbit anti-Ase (Brand et al., 1993 (link)) (1:200), guinea pig anti-Deadpan (provided by James Skeath, Department of Genetics, Washington University of St. Louis; 1:2000) and anti-Eyeless (Kammermeier et al., 2001 (link)) (1:200). Secondary antibodies were Alexa Fluor 405-, Alexa Fluor 555-, Alexa Fluor 633-, or Alexa Fluor 488-conjugated (Invitrogen, A-11039, A-21435, A-21105, A-21072, A-21094, A-21200) and used at 1:300, 1:500, 1:300 and 1:500, respectively. DNA stain was DAPI (Sigma). Samples were mounted in Vectashield. Quantification was performed using ImageJ and Imaris (Bitplane).

Vero E6 cells were cultured on coverslips in 24-well plates. Twenty-four hours post-seeding, cells were rinsed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 min and washed twice with PBS. Then, they were blocked with a solution containing 4% FBS in PBS for 30 min at room temperature, and incubated with 1:100-diluted anti-SERINC5 (ab204400), rabbit anti-MAVS (24,930, Cell Signaling) or mouse anti-MAVS (sc-166,583) in blocking solution for 1 h at room temperature. After three washes with PBS, bound antibodies were detected by incubation, as appropriate, with AlexaFluor 594-conjugated anti-mouse (A11020, Invitrogen) or AlexaFluor 633-conjugated anti-rabbit (A21072, Invitrogen) secondary antibodies in blocking solution for 1 h at 37°C. Slides were mounted in Prolong Gold antifade reagent with DAPI (Molecular Probes, 936,576) and images were obtained with an Apotome-equipped Axio Observer Z1 microscope (Carl Zeiss AG).