Figure 4

Effects of immunization of S-derived peptides on zebrafish larvae. (A) Kaplain-Meyer curve representing the survival rate (y-axis) over 10 days (x-axis) of larvae immunized with water, and with different concentration (1 or 10 µg/mL) of PSPD2002 and PSPD2003. (B) Table with the main histopathological changes observed in zebrafish larvae immunized with PSPD2002 and PSPD2003. Different organs from different systems were analyzed and classified according to the alterations/pathology in a score ranging from 0 to 10. (C) Representative images obtained in confocal microscope of transgenic Tg(mpeg:mCherry/tnfa:eGFP) larvae immunized by inoculation into the swim bladder with different concentrations of PSPD2002 and PSPD2003 at 1, 2 and 6 days post injection (dpi). Macrophages are marked by red fluorescence (mCherry) expression and tnfa by green fluorescent protein (GFP) expression. The region surrounded by the yellow dash represents the counting region. Lateral view in sagittal plane of the animal, with caudal-cranial orientation (left–right) and pelvic fin below, allowing visualization of the swim bladder (centered), intestinal tract (below), and notochord and muscle tissue above. Counting the total number of macrophages (mCherry+) (D) and double-labeled pro-inflammatory macrophages (mCherry + /tnfa +) (E) in larvae immunized with PSPD2002 at 1, 2 and 6dpi. Counting the total number of macrophages (mCherry +) (F) and dual labeled pro-inflammatory macrophages (mCherry + /tnfa +) (G) in larvae immunized with PSPD2003 at 1, 2 and 6dpi. Scale bar 100um in (C) From C to G, experiments were performed in three independent days and n vary from 4 to 12. All data are expressed as the mean ± standard deviation. *p < 0.05; **p < 0.01.