Rabbit anti activated caspase 3

The following antibodies were used for biochemistry experiments: mouse anti-β-actin (1:5000, Proteintech), mouse anti-Myc (1:1000, Millipore), rabbit anti-Pyk2 (1:500, Abcam). The following antibodies were used for immunocytochemistry and immunohistochemistry: mouse anti-Tuj1 (1:300, Covance), rabbit anti-αCD (1:500, Synaptic Systems), rabbit anti-GFP (1:1000, Invitrogen), rabbit anti-BLBP (Brain lipid binding protein) (1:500, Chemicon), mouse anti-GM130 (1:1000, BD Bioscience), rat anti-BrdU (1:1000, Bio-Rad), rabbit anti-activated caspase 3 (1:500; Cell Signaling Technology), rabbit anti-Tbr2 (1:500, Abcam), rabbit anti-WAVE2 (1:500, Millipore), goat anti-rabbit Alexa Fluor 488 (1:300, Molecular Probes), goat anti-rabbit Alexa Fluor 568 (1:300, Molecular Probes), goat anti-mouse Alexa Fluor 568 (1:300, Molecular Probes), goat anti-mouse Alexa Fluor 647 (1:300, Molecular Probes).

Embryos were incubated in egg water with 0.003% PTU to prevent pigment formation. Larvae were anesthetized in 0.02% tricaine and fixed in 4% PFA overnight. Samples were washed in 1× PBS followed by 1× PBST and treated with 20 µg/mL Proteinase K (New England Biolabs, Ipswich, MA, USA). The reaction was stopped by adding 10% lamb serum (Gibco, Dublin, Ireland) followed by additional washes in PBST. Samples were blocked with 10% lamb serum for 1–4 h and incubated in primary antibodies followed by secondary antibodies. Antibodies used included rabbit anti-GFP (1:100; Invitrogen, Carlsbad, CA, USA), rabbit anti-activated Caspase-3 (1:100; Cell Signaling Technology, Danvers, MA, USA), mouse anti-Zpr1 (1:50; ZIRC, Eugene, OR, USA), and mouse anti-Cldn5 (1:100; Invitrogen, Carlsbad, CA, USA), Alexa Fluor goat anti-rabbit 488 (1:200; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor goat anti-mouse 555 (1:200; Invitrogen, Carlsbad, CA, USA). Samples were imaged on Nikon C1Si laser scanning confocal microscope. Z-stacks were compiled to create maximum intensity projection images using Nikon NIS-Elements Version 3.1 imaging software.

FD and TA muscles were cut into 50 μm-thick longitudinal slices using a cryostat (Leica Microsystems^®, Wetzlar, Germany), collected on glass slides (Superfrost Plus Slat^®, Thermo Scientific^®, Waltham, MA, USA), rehydrated in a phosphate buffer solution (PBS), and then immersed in PBS containing 10% normal donkey serum (NDS) and 0.2% Triton X-100. Sections were then incubated overnight in the same blocking solution with a rabbit anti-activated caspase 3 (1/1000, Cell Signaling Technology^®, Beverly, MA, USA). Following several washes in PBS, sections were incubated for 2h in PBS containing 5% NDS with Alexa Fluor^® 488 donkey anti-rabbit antibody (1/400, Invitrogen^®, Carlsbad, CA, USA). Activated caspase 3 positive cells were quantified in muscles using an epifluorescence microscope (Leica Microsystems^®) to determine the apoptosis rate as the average number of dead cells per area of 0.3 cm².