Transgene Expression Verification in Zebrafish

To verify transgene expression, 7.5-SSL larvae were fixed in 4% paraformaldehyde, embedded in OCT (Tissue-Tek, VWR, Radnor, PA), and cryosectioned at 20 µm. Sections were washed with 0.3% Triton X-100 in PBS (PBSTX), blocked, then incubated at 4°C overnight with primary antibodies. Primary antibodies were mouse anti-PAX7 (Developmental Studies Hybridoma Bank) for the xanthophore lineage (Minchin and Hughes, 2008 (link)) and rabbit anti-GFP (ThermoFisher) for Venus. Notch signaling reporter Tg(TP1:H2B-mCherry) was visualized with rat anti-mCherry (ThermoFisher).

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Eom D.S., Bain E.J., Patterson L.B., Grout M.E, & Parichy D.M. (2015). Long-distance communication by specialized cellular projections during pigment pattern development and evolution. eLife, 4, e12401.

Publication 2015

mouse anti-PAX7 rabbit anti-GFP rat anti-mCherry

Antibodies Hybridoma Larvae Mouse Paraformaldehyde Pax7 Rabbit Tissue Transgene Triton x 100

Corresponding Organization :

Other organizations : University of Washington, Seattle University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Transgene expression

control variables

Paraformaldehyde concentration (4%)
Embedding medium (OCT)
Cryosection thickness (20 µm)
Washing solution (0.3% Triton X-100 in PBS)
Primary antibodies (mouse anti-PAX7, rabbit anti-GFP, rat anti-mCherry)
Incubation temperature (4°C)
Incubation duration (overnight)

positive controls

Tg(TP1:H2B-mCherry) reporter to visualize Notch signaling

negative controls

No negative controls explicitly mentioned

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