Fish gelatin

Planarians were equilibrated in PBSTx (5 min), then blocked O/N (16-20 hr) in BSA/fish gelatin blocking solution (0.6% IgG-free BSA (Jackson Immuno) and 0.45% fish gelatin (Sigma) in PBSTx) at RT. Supernatants were diluted 1:2 in blocking solution (i.e., one volume supernatant and one volume blocking buffer) in Figures 2 and 3, and Additional files 3, 4, and 5. In Figures 6 and 7, mAb 3F11 was diluted 1:2. In all other figures, supernatants were diluted 1:10 or 1:100 (2C11 only). Planarians were incubated O/N at 4°C. After 6-8 PBSTx washes over at least 6 hr, planarians were re-blocked for 1-2 hr at RT, then incubated with goat anti-mouse IgG + IgM HRP (Jackson Immuno) at 1:250 and DAPI (1 μg/ml) O/N at 4°C. For direct detection, goat anti-mouse IgG + IgM Dylight-488 (Jackson Immuno) was used at 1:500. After incubation in secondary antibody, animals were washed 6-8X in PBSTx over at least 6 hr, then twice in PBSTw (0.01% Tween-20 in 1X PBS) (5 min each). For TSA, planarians were incubated with FITC-Tyramide [34 (link)] at 1:1500 plus 0.005% H₂O₂ in PBSTw for 10-15 min at RT, then washed 3X in PBSTx (10 min each). Planarians were washed overnight in PBSTx, rinsed again in PBSTx, then mounted in Vectashield.