Immuno shot

Total protein was extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) from cultured cells. Following measurement of protein concentration (Protein Assay Rapid Kit Wako; Wako Pure Chemical Industries, Osaka, Japan), the total protein was individually subjected to SDS-PAGE (SuperSep Ace; Wako, Japan). These proteins were transferred onto Hybond P polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). Then the proteins on the membrane were blocked in 5% nonfat dry skim milk powder (Wako) for over 1 h at room temperature and were incubated with primary antibodies for 24 h at 4° C using Immuno Shot (Cosmo Bio, Tokyo, Japan). The dilutions of primary antibodies used in this study were as follows: p22^phox, 1:100;HIF-1α,1:200; E-Cadherin, 1:200; Vimentin, 1:200; β-actin, 1:1000. These antibody–protein complexes on the blot were detected using ECL-plus Western blotting detection reagents (GE Healthcare) following incubation with anti-rabbit or anti-mouse IgG horseradish peroxidase (GE Healthcare) at room temperature.

Chicken embryonic tissues (heart, retina, and cerebellum at E19) were dissected and snap-frozen in liquid nitrogen. Frozen tissues were ground up with a pestle in a chilled mortar and solubilized immediately in RIPA buffer [10 mM Tris-HCl (pH 7.4), 1% octylphenoxypolyethoxyethanol, 0.1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.15 M NaCl, 1 mM ethylenediaminetetraacetic acid]. Solubilized tissues were sonicated and centrifuged at 19000 ×g at 4°C for 15 min. The tissue lysate was subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto a polyvinylidene difluoride membrane, and probed with anti-cOpn3 or anti-cTMT-L antibody, both diluted 1:3,000 in Immuno Shot (Cosmo Bio Co. Ltd.; Tokyo, Japan). After washing, secondary antibody (anti-guinea pig IgG) was added at 1:10,000 dilution and signals were detected by with an ImmunoStar LD (Wako Pure Chemical Industries Ltd.; Osaka, Japan). To ascertain immunolabeling specificity, immunogen peptide preabsorption tests were performed with a solution in which the molar ratio of anti-cOpn3 or anti-TMT antibody and the indicated antigen peptides was 1:20.

Samples were soaked in xylene twice for 5 min to remove paraffin, and then 100% ethanol twice 3 min to hydrophilize. After washing with water in several minutes, the samples were soaked into epitope retrieval buffer (DAKO) and autoclaved at 105 °C for 10 min. The samples were then soaked into 1× PBS for several minutes and incubated with 100 μL of primary antibody in IMMUNO SHOT (Cosmo Bio) at 4 °C overnight. Antibodies and dilutions are shown in Supplementary Table 2. After two 5-min washes with PBS, the samples were incubated for 30 min at room temperature with secondary antibodies. The samples were washed twice in PBS for 5 min, and then nuclear staining was performed with hematoxylin for 10–30 s. After washing with water in 10 min, samples were placed in 100% ethanol (three times), and then placed in xylene three times. Finally, the samples were evaluated under a microscope (Keyence).