DOI: 10.7302/1hp2-hc61
title: Set of images for Figures 1 and 2 and 6 and Supplementary Figure 7

description: This dataset contains images of dissected and fixed retinae in which cones of specific subtypes are labeled either by transgenic expression of a fluorescent reporter or by antibody staining (Figures 1 and 2 and 6A and Supplementary Figure 7A).
This dataset also contains images of dissected and fixed retinae in ZO1 is immunostained (Figure 6C-E and Supplementary Figure 7B).
Please see the readme file for which files correspond to which figures.
description: Zebrafish:
Fish were maintained at approximately 28°C on a 14/10 h light/ dark cycle with standard husbandry procedures. Zebrafish lines, Tg(-5.5sws1: EGFP)kj9, Tg(-3.2sws2: mCherry)mi2007, Tg(trß2: tdTomato), Tg(-3.2sws2: EGFP), Tg(gnat2:H2A-CFP), and pigment mutant ruby carrying albino (slc45a)b4/b4 and roya9/a9 were used. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Michigan.
description: Histology:
Retinal dissection, fixation and immunocytochemistry were performed as previously described. Briefly, the isolated retina was fixed in 4% paraformaldehyde with 5% sucrose in 0.1M phosphate buffer, pH 7.4, at 4°C overnight. After antigen retrieval with 10 mM sodium citrate in 0.05% tween 20 (pH 6.0), the retina was incubated in blocking buffer for 2 hours followed by primary antibody incubation, mouse anti-Zonula Occludens (ZO1-1A1, 1:200, ThermoFisher Scientific, Waltham, MA) and rabbit anti-GFP (1:200, ThermoFisher Scientific) at room temperature overnight. Incubation with secondary antibodies (Alexa Fluor 555 and 649, ThermoFisher) was performed at room temperature overnight, and the retina was flat-mounted on a glass slide. For retinal cross sections, affinity-purified rabbit polyclonal opsin antibodies, a gift from Dr. David R. Hyde, were used. Images were acquired with a Zeiss AxioImage ZI Epifluorescent Microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with an ApoTome attachment for optical sectioning structured illumination, Leica DM6000 Upright Microscope System (Leica Microsystems, Werzlarm Germany) and a Leica TCS SP5 confocal microscope equipped with Leica 40X HCX PL APO CS Oil Immersion lens.
description: CRISPR-Cas9 mediated mutation in the thrb gene:
A genetic mutation targeting the type 2 isoform of the thrb gene (synonym, trß2) was generated by CRISPR-Cas9 gene editing methods. Briefly, pT7 gRNA vector (Addgene #46759) was used as a template to construct the thrb2 gRNA. PCR based method was performed using specific primers, 5’-GGGGTAATACGACTCACTATAGGCAACACAGCCAACCCTATGTTTTAGAGCTAGAAATAGCAAG-3’
description: 5’-AAAAAGCACCGACTCGGTGCC-3’. The MEGAscript T7 Kit (Ambion Inc., Austin, TX) was used to transcribe the gRNA. For the nlsCas9nl mRNA synthesis and purification, mMESSAGE mMACHINE T7 Transcription Kit (Ambion) and Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) were used. For genotyping of the thrb mutation, PCR fragments of the thrb gene, amplified using specific primer set, 5’-CATGGTGTAAGTGGCGGATATG -3’
description: 5’-TCCACTGCATCTGAGAGAAATCC-3’, were subjected to restriction with BstXI (New England Biolabs, Ipswich, MA).
description: Large tile scans of flat-mounted retinae:
Large tile scans of entire flat-mounted retinae from adult Tg(sws1:EGFP) zebrafish immunostained for ZO1 were acquired with a Leica TCS SP8 LSCM (Leica Microsystems) equipped with Leica 20X PL APO Dry lens. The GFP signal was recovered by immunostaining with anti-GFP antibody. The White Light Laser was tuned to 555 nm for Alexa Fluor 555 and 649 nm for Alexa Fluor 649. The Leica HyD hybrid detectors were tuned to 600-641 nm for Alexa Fluor 555 and 701-751 nm for Alexa Fluor 649. Images were acquired at 700 Hz scan speed with a resolution of 2048 x 2048 pixels in the xy dimension with a 2.0 um interval between optical sections in the z-dimension.