Ab125356

Fixed embryos were saturated with 30% sucrose in 1× PBS, cryo-sectioned in the coronal plane at the 12-µm thickness, and mounted on a slide glass. Antigen retrieval was performed in 10 mM sodium citrate with 0.05% Tween-20 (pH 6.0) at 95°C for 20 min. The slides were then blocked in 5% normal donkey serum in 1× PBS-T (0.05% Tween-20 in 1× PBS) for 30 min at room temperature and transferred to the blocking solution containing a rabbit anti-acetylated alpha tubulin antibody (1:300, ab125356; Abcam, UK). After incubating at 4°C for 16 h, the slides were washed three times with PBS-T, and the secondary antibody solution with an anti-rabbit IgG antibody conjugated to Alexa Fluor 555 (A-31572; Sigma) in PBS-T (1:1,000) was added. After incubating at room temperature for 1 h, the slides were washed three times with PBS-T, and nuclei were counterstained with Hoechst 33342 (Sigma) dissolved in 1× PBS (1:20,000). The slides were mounted in FluorSave (Sigma) with a cover glass. Five embryos were randomly chosen per group, and three coronal tissue sections around the longest axis of the eye were selected per embryo for cell counting in Fig. 3.