Gtx125851

Fixed specimens, both larvae at 15, 25, 32 dpf, and meiofauna-collected juveniles, were rinsed three times in PBS (pH 7.7) and then blocked for 2 hours in PBS with 6% bovine serum albumin (BSA) and 2% Triton X100 at RT. Renilla luciferase antibody (GTX125851, Genetex, [16 (link), 45 (link)]) was diluted 1:500 in PBS containing 6% BSA and 1% Triton X100. After overnight incubation (at room temperature), larvae were rinsed at least six times in PBS-1% Triton X100 and then incubated in a 1:500 dilution of Alexafluor 594 conjugated antirabbit (A11037, Thermofisher Invitrogen) in PBS containing 1% Triton X100 and 6% BSA. After incubation (2 hours, RT), larvae were rinsed six times with PBS-1% Triton X100. Specimens were mounted in Mowiol (Mowiol^® 4–88, Sigma) and examined using a Leica confocal microscope. For each stage, immunodetection experiments were performed on at least three specimens. A positive control experiment was performed using the same protocol on adult A. filiformis arms (S2 Fig). Besides, a control was performed by omitting the primary antibody to ensure the absence of unspecific binding of the secondary antibody.

Pyrosomes were fixed in 4% paraformaldehyde in PBS for 20 min and then incubated in 0.5% Triton in PBS for 1 h at room temperature on a rocking nutator. Samples were then blocked in PBT-BSA (0.5% Triton X-100, 0.5% bovine serum albumin) for 30 min at room temperature. An anti-Renilla luciferase antibody (1:250, GTX125851, GeneTex) or rabbit pre-immune serum (1:250, ab37415, Abcam) was added and samples were incubated at 4 °C overnight. GTX 125851 is a polyclonal antibody with specificity for Renilla luciferase. Samples were then washed in PBS and incubated with AlexaFluor-488-conjugated anti-Rabbit secondary (1:200, Invitrogen) for 1 h at room temperature. Following secondary antibody incubation, samples were washed in PBS and Hoechst 33342 (Molecular Probes, 1:2000) was added during a 10 min wash in PBS. Samples were dissected and placed in a glass-bottomed petri dish with PBS for imaging. In addition to the pre-immune serum control, other control samples were incubated without primary or secondary antibody, and no specific signal was observed (data not shown).