Additional Notesab13847 staining Salvelinus alpinus (a member of Salmonidae) by whole-mount assay in a pre-hatching embryonic stage. The embryos fixed in 4% paraformaldehyde were kept in 100% methanol at -20¯C and then rehydrated by increasing dilution series of 1 ¿ PBST (1 ¿ PBS with 0.1% Tween). The embryos were dechorinated and treated with 20 to 40 ´g/ml proteinase K (New England Biolabs) for 22 to 60 min, depending on the relative-ages. Prior to the blocking step embryos were washed with PDT buffer (1 ¿ PBST, 0.3% Triton X, 1% DMSO) for 1 hour at room temperature and then were transferred to blocking solution (10% heat-inactivated FBS, 8% BSA in PBST) for another hour at room temperature. A 1:500 dilution of anti-activated + pro Caspase-3 antibody (abcam, ab13847) was added and embryos were incubated at 4¯C overnight. A goat polyclonal secondary antibody to rabbit IgG - H&L (Alexa Fluor 488) (abcam, ab150077) was used for detection at 1:400 dilution and 4¯C overnight incubation. (A) Negative control with only the secondary antibody and (B) ab13847 stained embryo (Mo: mouth opening L: lower jaw).