Zebrafish Whole Mount In Situ Hybridization

Whole mount in situ hybridization (WISH) was conducted as described^{101 (link),102 (link)}. Anti-sense RNA probes were digoxigenin-labeled (etv5a, odf3b, cetn4, scl, irx2a, cdh17, slc20a1a, trmp7, slc12a1, slc12a3, jag2b) or fluorescein-labeled (smyhc1, cdh17, pax2a), and generated by in vitro transcription using plasmid or PCR templates as described^{16 (link),17 (link),36 (link),63 (link),103 (link)}. Embryos were mounted in glycerol and images were taken using a Nikon Eclipse Ni with a DS-Fi2 camera. Whole mount fluorescent in situ hybridization (FISH) was performed as described^{103 (link)} with the digoxigenin and fluorescein-labeled RNA probes used in WISH. Stains were developed with the TSA plus Cy3 and TSA plus Fluorescein kits (Perkin Elmer). Embryos were mounted in Poly Aqua-mount as described^{104 (link)} and imaged on a Nikon C2 confocal microscope. Z-stacks were processed with FIJI into max image projections, and all figures were assembled using Adobe Photoshop CS5.

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Marra A.N., Cheng C.N., Adeeb B., Addiego A., Wesselman H.M., Chambers B.E., Chambers J.M, & Wingert R.A. (2019). Iroquois transcription factor irx2a is required for multiciliated and transporter cell fate decisions during zebrafish pronephros development. Scientific Reports, 9, 6454.

Publication 2019

DS-Fi2 camera TSA plus Cy3 Nikon C2 confocal microscope

Cdh17 Confocal microscope Digoxigenin Embryos Fluorescein Fluorescent in situ hybridization Glycerol In situ hybridization Plasmid Poly Rna probes Stains Transcription

Corresponding Organization :

Other organizations : University of Notre Dame

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Variable analysis

independent variables

Anti-sense RNA probes used for in situ hybridization (etv5a, odf3b, cetn4, scl, irx2a, cdh17, slc20a1a, trmp7, slc12a1, slc12a3, jag2b, smyhc1, cdh17, pax2a)

dependent variables

Gene expression patterns visualized by whole mount in situ hybridization (WISH) and whole mount fluorescent in situ hybridization (FISH)

control variables

Protocol and methods used for WISH and FISH, as described in the referenced publications
Mounting of embryos in glycerol for WISH and Poly Aqua-mount for FISH
Imaging using Nikon Eclipse Ni and Nikon C2 confocal microscope

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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