Polyclonal antibodies against

The ED1 antibody reacted against extrinsic macrophages and intrinsic microglia^{28 (link),30 (link),31 (link)}. Monoclonal antibodies against ED1 (1:100, Abcam, Cambridge, MA, USA) were used. Arg1 and CD206 are M2 macrophage markers^{32 (link)}. Polyclonal antibodies against Arg1 and CD206 (1:100, Abcam, Cambridge, MA, USA) were used to determine macrophage polarization. The samples were incubated in a primary antibody overnight at 4 °C. The secondary antibody conjugated with fluorescein isothiocyanate (FITC and rhodamine, 1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was incubated at room temperature for 1 h. Counterstaining was performed using DAPI (1:1000, Sigma, St. Louis, MO, USA). For comparison, the ED1-positive cells were calculated in six HPFs (×400 magnification) at the optic nerve lesion site.

Detection of PLIN2, PLIN3, and PPAR-γ and β/δ in MT-III-stimulated VSMCs was performed by immunostaining. Cells were fixed in 2% PFA, permeabilized with 0.2% Triton-X 100 in 0.1 M phosphate buffer, and blocked with 0.5% albumin in 0.1 M phosphate buffer for 30 min. After PBS washes, VSMCs were incubated for 1 h with polyclonal antibodies against PLIN2 and PLIN3 (Abcam, CA, USA) or PPAR-γ and β/δ (1:250) (Santa Cruz, TX, USA) diluted in 0.1 M phosphate buffer with 0.2% Triton-X 100. After three washes with PBS, the preparations were incubated with secondary Alexa488-conjugated antibody (1:500) (Thermo Fisher, Waltham, MA, USA) for 1 h. After the washes, the slides were mounted with Fluoromount-G and examined under a confocal laser scanning microscope (Zeiss LSM 510 Meta).

Monoclonal antibodies against DDHD1 (12D10) were raised previously (Baba et al., 2014 (link)). Polyclonal antibodies against syndapin2 and α-tubulin were obtained from Abgent and Sigma-Aldrich, respectively. Polyclonal antibodies against MICAL-L1 and Rab13 were purchased from Abcam. Monoclonal antibodies against MICAL-L1, EEA1, and TfR were obtained from Abnova, BD Transduction Laboratories, and Sigma-Aldrich, respectively. Alexa Fluor 488-conjugated goat anti-mouse antibodies and Alexa Fluor 594 goat anti-mouse antibodies were purchased from Invitrogen. The following reagents were used: NGF (Alomone Labs); RA, unlabeled-Tfn, deferoxamine, R59949, FITC-phalloidin and TRITC-phalloidin (Sigma-Aldrich); CAY1059 and CAY10594 (Cayman Chemical); Alexa488-Tfn and TRITC-Tfn (Thermo Fisher Scientific); and EGF (Invitrogen).

The shrimp hemocytes were lysed in RIPA lysis buffer (Beyotime, China) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) on ice. The protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and 20 μg of protein was used for each sample. After separation on a 15% SDS-polyacrylamide gel, the proteins were electrotransferred to a nitrocellulose membrane (GE Healthcare, Germany) in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol). The membrane was blocked with 5% nonfat milk in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) for 2 h at room temperature. Subsequently, the membrane was incubated with a primary antibody in TBST buffer containing 1% nonfat milk overnight at 4°C. After extensive washing in TBST buffer, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Bio-Rad, USA) for 2 h at room temperature. The membrane was detected by using a Western Lightning Plus-ECL kit (Perkin Elmer, USA). The monoclonal antibody against Fas was purchased from Cell Signaling Technology (USA). The polyclonal antibodies against PF4 and IL-22 were purchased from Abcam (USA).