Jb 4 plastic resin

Single-color, whole-mount in situ hybridization was performed as previously described (Xu et al., 2002 (link)). Cilia, basal body, and apical-basal polarization of KV cells were visualized using antibodies against acetylated α-tubulin (Sigma-Aldrich), γ-tubulin (Sigma-Aldrich), and the tight junction protein ZO-1 (Invitrogen) as previously described (Lin and Xu, 2009 (link)). Cilia length and number were measured using AxioVision software (Zeiss) and analyzed by the Student t-test. For analysis of the ectopic protrusion, immunostaining was performed in whole-mount embryos, and then the ectopic protrusion was dissected and mounted in VECTASHIELD (Vector Laboratories), or the embryos were embedded in JB-4 plastic resin (Polysciences) and sectioned at 4-μm sections (Sullivan-Brown et al., 2011 (link)).

Dehydrated embryos or adult eyes were embedded in JB-4 plastic resin (Polysciences) and then sectioned (5 μm) using a LEICA RM 2165 rotary microtome. Sections, in which optic nerve was present, were stained with Hoechst (H3570, 1:2000, Thermo Fisher Scientific) and BODIPY TR Methyl Ester (C34556, 1:200, Thermo Fisher Scientific), and mounted using Permount (SP15-500; Thermo Fisher Scientific). Two-color z series were acquired using a Zeiss LSM5 Pascal confocal microscope via sequential laser excitation with 40× and 63× oil objectives. Images were deconvolved using Huygens Essential software and processed using Adobe Photoshop. Adult eye sections were stained with Methylene Blue/Azure II (Humphrey and Pittman, 1974 (link)) and examined using a Nikon E800 microscope equipped with a Spot Insight charged-couple device digital camera. Quantification of photoreceptor outer segment (OS) length was performed on maximum intensity z projections of confocal stacks created in ImageJ. OSs were traced and measured using the freehand line tool.