Rabbit anti mouse ki67 antibody

GFP and Tomato signals were detected without immunofluorescence staining. Cryosections were dried at room temperature for 30 minutes, rehydrated in PBS for 10 minutes, and then mounted with Fluoromount (eBioscience, Santa Clara, CA, USA; ref 495952).
For Ki67 immunostaining, tissue sections were incubated in PBS supplemented with 5% normal goat serum for 30 minutes and with rabbit anti‐mouse Ki67 antibody (dilution 1/200; Abcam, Cambridge, MA, USA; ref ab15580) overnight at 4°C. Secondary goat anti‐rabbit AF647 antibody (Life Technologies; ref A‐21245) was incubated for 1 hour at room temperature and slides were mounted with Fluoromount (ref 495952, eBioscience). For phospho‐Smad1/5/9 immunostaining, citrate buffer antigen retrieval was used for 20 minutes at 95°C followed by 20 minutes at 4°C. Sections were incubated in PBS supplemented with 5% normal goat serum for 30 minutes, before incubation with rabbit anti‐mouse phoshoSmad1/5/9 antibody (dilution 1/200; Cell Signaling Technology, Danvers, MA, USA; ref 13820 T) overnight at 4°C, and with secondary goat anti‐rabbit AF647 antibody for 1 hour at room temperature. Slides were mounted with Fluoromount and images were acquired using a Zeiss LSM 800 confocal microscope.

Materials were purchased from standard sources: DOTAP (Avanti Polar Lipids Inc., Alabaster, AL, USA); MTT (MilliporeSigma Co., St. Louis, MO, USA); DMEM and fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA); methanol and acetic acid (high performance liquid chromatography grade; Thermo Fisher Scientific, Waltham, MA, USA); and DMSO and acetone (KeLong Chemicals, Chengdu, Sichuan, People’s Republic of China). Antibodies used included rabbit anti-mouse Ki67 antibody and rhodamine-conjugated secondary antibody (both from Abcam, USA).
The MPEG (2000)-PLA(4000) diblock copolymer with a designed molecular weight of 3,000 Da was synthesized through the opening of the l-lactide ring and initiated by MPEG. The MPEG (5.0 g) was melted in a 70-mL flask following the addition of Sn(Oct)2 (1 mL) and anhydrous l-lactide (10 g) under nitrogen. The reactant mixture was maintained for 1 day at 125°C. The crude product was dissolved in tetrahydrofuran and then purified by precipitation in ice-cold diethyl ether, followed by filtration. Finally, the product was vacuum dried at ambient temperature. The average molecular weight number (Mn) of the MPEG-PLA copolymer was 6,010 Da (data not shown). MPEG (with a molecular weight of 2,000 Da; Sigma-Aldrich Co.) was dried in a one-necked flask under vacuum and stirred at 105°C for 90 min before use.