Mouse anti gfp clones 7 1 and 13

Adult Ciona robusta (intestinalis Type A) were collected by M-REP (San Diego, USA). Gametes were isolated for in vitro fertilization and dechorionation and subsequent electroporation following standard protocols 53, 54 . All plasmid sequences and mixes are described in Supplemental File 5. Embryos were raised at 20°C and fixed at the desired stage as calculated by hours post-fertilization. To obtain juveniles, larvae were allowed to metamorphose on (but not attach to) agarose-coated petri dishes in filtered/buffered artificial sea water supplemented with 1X penicillin-streptomycin (Omega Scientific, catalog number PS-20), followed by daily changes of penicillin-streptomycin sea water. For direct visualization of fluorescent proteins, in MEM-FA (3.7% formaldehyde, 0.1M MOPS pH7.4, 0.5M NaCl, 1 mM EGTA, 2 mM MgSO4, 0.05% Triton-X100) for 15 minutes, rinsed in 1X PBS/0.4% Triton-X100/50mM NH4Cl and 1X PBS/0.05% Triton-X100. For immunostaining of CD4::GFP, embryos were fixed and rinsed as above and incubated in mouse anti-GFP (clones 7.1 and 13.1, Roche) at 1:500 dilution for 1 hour, and AlexaFluor 488 goat anti-mouse IgG secondary (ThermoFisher, catalog number A11001) at 1:500 dilution for 1 hour. Both incubations were done in 1X PBS/0.05% Triton-X100/2% Normalized Goat Serum and rinsed in 1X PBS/0.05% Triton-X100. All samples were mounted in 1X PBS/50% Glycerol/2% DABCO.