Whole-mount immunostaining was performed as described previously69 (link), with the following modification: Larvae were fixed in 4% PFA after stopping the heart with 0.2% Tricaine to prevent cardiac collapse during fixation. The primary antibodies used in this study are Mouse anti-MHC (MF20) (1:500, Invitrogen 14-6503-82), Mouse anti-Alcama (1:500, ZN-8, DSHB), and Rabbit anti-Fli1 (1:500, Abcam ab133485), followed by the secondary antibodies: Goat anti-Mouse-Alexa568 (1:500, Invitrogen A11004), Goat anti-Mouse-Alexa647 (1:500, Invitrogen A21236), and Goat anti-Rabbit-Alexa488 (1:500, Invitrogen A110034). All images were analyzed using an LSM 700 confocal laser scanning microscope (Zeiss) with a 40x objective, except for the samples shown in Fig. 2t, u, which were imaged using an LSM 800 Examiner confocal laser scanning microscope (Zeiss) with a 40x objective. For AV cell number quantification, the cells were counted in every plane of a z-stack covering the AV canal. The 78 hpf wild-type dataset (n = 61) is the same in Figs. 1n, 2s, 3o, and represents a pool of pkd1a+/+, pkd1a+/+; pkd2+/+, and camk2g1+/+; camk2g2+/+ larvae. The 78 hpf pkd1a mutant dataset (n = 26) is the same in Figs. 1n, 2s, 3o.
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