Alexa fluor 594 antimouse cd31 antibody

AP1 peptide was purchased from Sangon Biotech Co. (Shanghai; HPLC-purified; purity > 95%, identified by MS). Sodium cholate was from Shanghai Chemical Reagent Company. Methoxy-poly (ethylene glycol)-poly (lactic acid) (MPEGePLA, Mw 33,000 Da) and R-carboxyl-poly (ethylene glycol)-poly (lactic acid) (COOH-PEG-PLA, Mw 33,400 Da) block copolymers were purchased from Adamas Corporation (Shanghai local agent, China). Doxorubicin (DOX) was obtained from Beijing Huafeng United Technology (China). DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethy-lindotricarbocyanine iodide), a near-infrared dye used for the evaluation of in vivo targeting assay, was obtained from Biotium (Invitrogen, USA). Alexa Fluor 594 antimouse CD31 antibody was purchased from Biolegend. Dulbecco's modified Eagle medium (DMEM; high glucose) cell culture medium, certified fetal bovine serum (FBS), penicillin/streptomycin stock solutions, and 0.25% Trypsin-EDTA were purchased from Gibco (USA). All other chemicals, if not indicated specifically, were obtained from Sigma-Aldrich.

Cells cultured in the channels were immunostained for CD41 and CD31, which are a transient marker of fetal HSC and an adhesion molecule expressed on endothelial cells, respectively. Briefly, cells were washed with 60 µL PBS(+) three times for 2 min each, fixed with 60 µL 4% paraformaldehyde at 4 °C for 10 min, washed with 60 µL PBS(+) (3 × 2 min washes), and then incubated with 60 µL of 1:250 dilution of Alexa Fluor 488 anti-mouse CD41 antibody (133908, BioLegend) and Alexa Fluor 594 anti-mouse CD31 antibody (102520, BioLegend) in PBS, for 2 h at 4 °C. The cells were then washed with PBS(+) three times for 2 min each.

Frozen tissue sections were prepared and covered with OCT media. Then the liver tissue sections were fixed with zinc fixative solutions for 10 min. After rinsing slides with PBS for 3 times, the liver tissues were further treated with 2% Triton X-100 for 15 min. Then 100 µL blocking buffer (10% FBS in PBS) was added onto tissues for 1 h. Antibodies including F4/80 Monoclonal Antibody (BM8), FITC (Thermo Fisher Scientific, #11-4801-85, 1:100), Alexa Fluor® 594 anti-mouse CD31 Antibody (Biolegend, #102520, 1:100), anti-mouse Ly6G/Ly6C (Gr-1) (BioXcel, #BE0075, 1:200) and Anti-Caspase-3 antibody (Abcam, #ab13847, 1:200) were diluted and added on slides and incubated for overnight at 4 °C. To stain the secondary antibody, slides were washed and incubated with secondary antibody conjugated with Goat Anti-Rabbit IgG H&L (Alexa Fluor 594) (Abcam, #ab150080, 1:200) or Goat Anti-Rat IgG H&L (Alexa Fluor 488) (Abcam, #ab150157, 1:200) for 1 h. After that, the slides were rinsed with PBS and mounted with DAPI-containing mounting solution. Fluorescence images were acquired via confocal microscopy.