Dual-labeling of Elavl1 and GFP in Chick Embryos

Embryos were electroporated bilaterally with MCP-GFP alone (‘control’), or MCP-GFP and MS2-Draxin 3′-UTR (‘experimental’), grown to stage HH9, then fixed at room temperature for 1 hr, washed, embedded, and cryosectioned as described (Hutchins and Bronner, 2018 (link); Hutchins and Bronner, 2019 (link)). Sections were processed using a DuoLink PLA kit (Millipore/Sigma) with Anti-Rabbit MINUS PLA probe, anti-Goat PLUS PLA probe, and Far Red PLA detection reagent, and primary antibodies for Elavl1 and GFP, according to the manufacturer’s DuoLink PLA Fluorescence protocol.

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Hutchins E.J., Gandhi S., Chacon J., Piacentino M, & Bronner M.E. (2022). RNA-binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification. eLife, 11, e63600.

Publication 2022

DuoLink PLA kit DuoLink PLA

Antibodies Embryos Fluorescence Goat Rabbit

Corresponding Organization :

Other organizations : California Institute of Technology, University of California, San Francisco, Miller College, University of California, Berkeley, California State University, Northridge

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Variable analysis

independent variables

Electroporation treatment (control vs. experimental)

dependent variables

Localization and/or expression of Draxin mRNA (as detected by the MS2-Draxin 3'-UTR reporter)

control variables

Developmental stage (HH9)
Sample preparation (fixed, washed, embedded, cryosectioned)
Detection method (DuoLink PLA with anti-Elavl1 and anti-GFP antibodies)

positive controls

Embryos electroporated with MCP-GFP alone ('control')

negative controls

None explicitly mentioned

Annotations

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