Tubulin tracker green

The paclitaxel was purchased from Wuxi Taxus Pharmaceutical Co., Ltd. (Jiangsu, China). Succinic anhydride (SA), 4-dimethyl aminopyridine (DMAP), 1-hydroxy-benzotriazole (HoBt), and 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Macklin (Shanghai, China). N,N-Diisopropylethylamine (DIPEA) was purchased from Aladdin (Shanghai, China). N-(2-Aminoethyl) maleimide trifluoroacetate salt (MAL) was purchased from J&K Chemical Ltd. (Beijing, China). TAR peptide (CRKKRRQRRRATWLPPR) was synthesized from China Peptide Co., Ltd. (Shanghai, China). Esterase and coumarin-6 were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China) Rabbit anti-NRP-1 antibody was purchased from Abcam (Cambridge, UK). Rabbit anti-β-actin antibody and secondary antibody (anti-rabbit) were purchased from ProteinTech (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from BioFroxx (Einhausen, Germany). Cy5.5 maleimide (non-sulfonated, Cy5.5-MAL) was obtained from APExBIO (Houston, TX, USA). Tubulin-tracker green and DAPI were obtained from Beyotime (Shanghai, China). Regular agarose G-10 was purchased from Biowest (Spain). F-12K medium, heparin and endothelial cell growth supplement (ECGS) were obtained from MacGene (Beijing, China). 1640 medium was obtained from Meilunbio (Dalian, China).

Frozen tissue sections (8umm thick) or cell sheets were fixed using a 4% paraformaldehyde solution, followed by permeabilization using 0.2% Triton X-100 for 10 minutes. Subsequently, blocking was performed using a 5% bovine serum albumin (BSA) solution from Sigma for a duration of 2 hours at room temperature. The primary antibodies were incubated overnight at 4 °C, followed by treatment with a fluorescein-conjugated anti-rabbit secondary antibody and Tubulin Tracker Green (Beyotime Biotechnology, Shanghai, China) were incubated for 1 hour at room temperature. After a 10-minute incubation period with DAPI, the fluorescence signal was visualized using either an Olympus IX70 fluorescence microscope (Olympus, Japan) or a confocal imaging system (Zeiss, Germany).

Polydimethylsiloxane (PDMS) pre-polymer (SYLGARD™ 184) was purchased from Dow (Midland, MI, USA); 97% Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane (FOTS) was purchased from J&K Scientific (Beijing, China); 99.99% Tetrahydrofuran (THF) and 99.8% dimethylformamide (DMF) were purchased from Merck (Rahway, NJ, USA); 99.5% Paclitaxel was purchased from Xi’an Haoxuan Biological Technology Co., Ltd. (Xi’an, China). PEGylated (methoxypolyethylene glycol 2000) paclitaxel (PEG-PTX) was synthesized according to the previous literature [25 (link),26 (link),31 (link)]. Tubulin-tracker green and thiazolyl blue tetrazolium bromide (MTT) assays were purchased from Beyotime (Nantong, China).