Immunofluorescence and Western Blot Protocols

For immunofluorescence analysis we used the following antibodies: sheep anti-Cnn (1:500) (Cottee et al., 2013 (link)); rabbit anti-PLP (PPHA) (1:500) (Martinez-Campos et al., 2004 (link)); mouse anti-TACC (1:500) (Gergely et al., 2000 (link)); rat anti-Asl (1:500) and rat anti-Spd2 (1:500) (Baumbach et al., 2015 (link)). Secondary antibodies were from Molecular Probes (Life Technologies): Alexa Fluor 405, 488 and 592 (all used at 1:1000). To enhance GFP fluorescence for 3D-SIM on fixed embryos we used GFP-Booster ATTO488 1:500 (Chromotek). For western blotting rabbit anti-PLP (PPHA) (1:1000) (Martinez-Campos et al., 2004 (link)) and mouse anti-actin (1:1000) (Sigma) were used. Secondary antibodies conjugated to IRDye 680 and IRDye800 were used at a concentration of 1:10,000 and blots imaged on an Odyssey CLx imager (LI-COR).