Alexa 568 goat anti rabbit igg secondary antibody

Zebrafish whole-mount immunofluorescence was performed as previously reported^{63 (link)}. Briefly, 2-dpf embryos were fixed in 4% paraformaldehyde at 4 °C overnight. Then embryos were washed 4 × 5 min in PBST (PBS + 0.5% Triton X-100) and incubated in a blocking solution (PBST + 10% normal goat serum + 1% BSA) for 2 h at room temperature. Embryos were then incubated with a rabbit anti-Cdh5 primary antibody (1:500, from Dr M Affolter's Lab) in the blocking solution at 4 °C overnight. Embryos were further washed 6 × 30 min in PBST and then incubated with an Alexa-568 goat anti-rabbit IgG secondary antibody (1:1000, Invitrogen) in the blocking solution at 4 °C overnight. After washing 3-5 times in PBST, images were taken with an Olympus Fluoview 1000 confocal microscope (Olympus, Japan).

Lipopolysaccharide (LPS) from Escherichia coli O55:B5 was purchased from Sigma-Aldrich (St Louis, MO, USA) and agomelatine from Aladdin (Shanghai, China). The monoclonal rabbit anti-LC3 (12741), anti-mTOR (2972), anti-Beclin-1 (3495), anti-p62 (23214), anti-ERK (9102), anti‐phospho‐JNK (4668), anti‐phospho‐ASK1 (3764), anti‐phospho‐p38 (9211), anti-Cleaved Caspase3 (9661), anti-Doublecortin (4604) and anti-β-actin (4970) were all purchased from Cell Signaling Technology. Anti-Gαi-2 (sc-13534) was purchased from Santa Cruz Biotechnology, anti-NF-κB P65 (bs90940), anti-phospho-PKA (bs4345) from Bioworld and anti-5-HT2C Receptor (DF3501) from Affinity Biosciences. Polyclonal rabbit anti-ionized calcium binding adaptor molecule-1 (Iba-1) (019-19741) was purchased from Wako Pure Chemical Inc. and Hoechst 33258 (C0031) from Solarbio. The polyclonal goat anti‐rabbit secondary antibody and the polyclonal goat anti‐mouse secondary antibody were purchased from Beijing Zhongshan Golden Bridge. Alexa-568 goat anti-rabbit IgG secondary antibody was purchased from Invitrogen and Alexa-488 goat anti-mouse IgG secondary antibody from Abcam Plc.

Cells were fixed with 2% formaldehyde solution for 15 min at room temperature and then permeabilized in ethanol 70% overnight at 4 °C. After 3 washing with PBS–Tween 0.02%, cells were saturated in PBS–BSA 2% for 10 min at room temperature. During 4 h, cells were incubated with the following primary antibodies (1/100e): mouse control IgG (Cell Signaling, 3900S), rabbit control IgG (Cell Signaling, 3900S), rabbit anti-EZH2 IgG (Cell Signaling) and mouse anti-Ki67 IgG1 (Cell Signaling, 9449S). After 3 washing with PBS–Tween 0.02%, cells were saturated in PBS–BSA 2% for 10 min at room temperature. Cells were incubated with the following secondary antibodies (1/200e): goat anti-Mouse IgG Alexa 488 (Invitrogen, A11001) or Goat anti-Rabbit IgG secondary antibody Alexa 568 (Invitrogen, A11011). Cells were washed 3 times with PBS–Tween 0.02% and covered with Prolong Diamond with 4',6-diamidino-2-phenylindole (DAPI).