Polyclonal anti laminin l9393

Immunostaining on whole-mount larvae and 140 μm vibratome sections were performed as described (Field et al., 2003 (link); Trinh and Stainier, 2004 (link)). We used the following antibodies: chicken anti-GFP (GFP-1020; Aves Labs; Tigard, OR) at 1:1000, polyclonal anti-laminin (L9393; Sigma-Aldrich, St Louis, MO) at 1:100, and goat anti-chicken 488 (A-11039; Life Technologies, Carlsbad, CA) at 1:200. To analyze HSC proliferation during ZM306416 treatment, Tg(hand2:EGFP) larvae were exposed to 2% ethanol for 24 h, allowed to recover in embryo medium for 3 h, and incubated in 7 µM EdU along with 3 µM ZM306416 for another 24 h. Proliferating cells were detected using Click-iT EdU Imaging Kit (Life Technologies). The TUNEL reaction was performed on 140 μm vibratome sections using ApopTag^® Red In Situ Apoptosis Detection Kit as described by the manufacturer's instructions (EMD Millipore, Billerica, MA). The samples were imaged on a Nikon A1Rsi inverted confocal microscope (Nikon Instruments, Melville, NY) at the Confocal Imaging Core at CCHMC. Image processing and quantification were conducted using Imaris software (Bitplane, Concord, MA).