Immunolocalization and Western blot of Ttpa protein in rat liver and zebrafish embryos (c269, RedEfsh and Casper). (A) The 5D strain fish expressed Ttpa in olfactory pits, gill arches and gut lining at 5 dpf. Anti-Ttpa was used to detect and validate Ttpa protein expression in 5D embryos initially to determine regionalization followed by in vivo observation. Figure A panels were generated with the BZ-x700 microscope, processed with BZ-X Analyzer Software with image adjustments equally applied across all images in Adobe Photoshop. All images are taken at 2× magnification at the same exposure protocol. This figure was created with Adobe Photoshop, v21.2.1. (B) The same anti-Ttp antibody, used in a Western blot, confirmed that Ttpa expression is not affected by the insertion of the mScarlet coding sequence in RedEfish relative to Casper or the c269 transgenic lines. This antibody reacts poorly with rat liver protein. RFP was also detected in the RedEfish embryos, although not detected by blot in the rat liver, c269 or Casper zebrafish embryos. (C) Shown are α- and γ-tocopherol concentrations (mean ± standard deviation, pmol/embryo, * = Student’s t-test, p = 0.002 and p = 0.001, respectively) in 5 day old Casper (hatched bars) and RedEfish (red solid bars) embryos (n = 5 homogenate pools per group, n = 15 embryos per pool). Both RedEfish and Casper embryos contained lower mean γ- as compared with α-tocopherol concentrations; the comparison of logarithmically transformed data for each fish line was significantly different (p < 0.0001, Holm-Sidak’s multiple comparisons test).
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