Zebrafish Embryo Analysis by In Situ Hybridization

Zebrafish embryos were fixed with 4% paraformaldehyde and analyzed by whole mount in situ hybridization using the protocol described previously[44 (link)]. The following antisense riboprobes were generated in this study: spaw[45 (link)], myl7[46 (link)] and foxa3[47 (link)]. Whole mount immunofluorescence was performed to detect monocilia in Kupffer’s vesicle as described[48 (link)]. Acetylated tubulin monoclonal antibody (1:1000; Sigma) and Cy3-conjugated anti mouse IgG antibody (1:500; Jackson ImmunoResearch Laboratories) were used in this study. Images were acquired on a Zeiss LSM700 confocal microscope. Images were processed using ImageJ software (NIH).

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Li Y., Yagi H., Onuoha E.O., Damerla R.R., Francis R., Furutani Y., Tariq M., King S.M., Hendricks G., Cui C., Saydmohammed M., Lee D.M., Zahid M., Sami I., Leatherbury L., Pazour G.J., Ware S.M., Nakanishi T., Goldmuntz E., Tsang M, & Lo C.W. (2016). DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia. PLoS Genetics, 12(2), e1005821.

Publication 2016

Acetylated tubulin monoclonal antibody Cy3-conjugated anti mouse IgG antibody LSM700 confocal microscope

Anti antibody Confocal microscope Embryos Immunofluorescence In situ hybridization Monoclonal antibody Mouse Paraformaldehyde Tubulin Zebrafish

Corresponding Organization : University of Pittsburgh

Other organizations : Tokyo Women's Medical University, Indiana University – Purdue University Indianapolis, UConn Health, University of Massachusetts Chan Medical School, Children's National, Children's Hospital of Philadelphia, University of Pennsylvania

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Variable analysis

independent variables

Paraformaldehyde concentration (4%)
Antisense riboprobes: spaw, myl7, foxa3

dependent variables

Expression patterns of spaw, myl7, foxa3 genes
Presence and distribution of monocilia in Kupffer's vesicle

control variables

Whole mount in situ hybridization protocol
Whole mount immunofluorescence protocol
Antibodies: Acetylated tubulin monoclonal antibody, Cy3-conjugated anti mouse IgG antibody
Imaging method: Zeiss LSM700 confocal microscope
Image processing software: ImageJ (NIH)

controls

Positive control: Not specified
Negative control: Not specified

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