Mouse anti cldn5

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.

Embryos were incubated in egg water with 0.003% PTU to prevent pigment formation. At 4 and 6 days post-fertilization (dpf), larvae were anesthetized in 0.02% tricaine and fixed in 4% PFA overnight. The next day, samples were washed in 1× PBS followed by 1× PBST and treated with 20 μg/mL Proteinase K (New England Biolabs) for 15 min. The reaction was stopped by adding 10% lamb serum (Gibco) followed by additional washes in PBST. Samples were blocked with 10% lamb serum for 1–4 h and incubated in primary antibody at 4° overnight. Samples were washed the next day in PBST and incubated in secondary antibody at 4° overnight. Antibodies used included rabbit anti-GFP (1:100; Invitrogen), mouse anti-Zpr1 (1:50; ZIRC), mouse anti-Cldn5 (1:100; Invitrogen), Alexa Fluor goat anti-rabbit 488 (1:200; Invitrogen), and Alexa Fluor goat anti-mouse 555 (1:200; Invitrogen). Additional washes in PBST were done the following day and stored in 1× PBS. Samples were embedded in 0.8% low-melting point agarose (Invitrogen) made in 1× PBS and imaged on Nikon C1Si laser scanning confocal microscope. Z-stacks were compiled to create maximum intensity projection images using Nikon NIS-Elements 3.1 imaging software.