Hif 1α antibody

Human hemoglobin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 3-(4,5-dimethy lthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, and hoechst 33342 were purchased from Sigma (St Louis, MO). 4-Dimethylaminopyridine (DMAP) and ODA were acquired from Aladdin Inc. (Shanghai, China). Singlet oxygen green reagent (SOSG) was acquired from Invitrogen Corp. (Carlsbad, CA). ICG was supplied by TCI (Tokyo, Japan). Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000), egg phosphatidyl lipid-80 (E80), and cholesterol were purchased from Lipoid GmbH (Ludwigshafen, Germany). ROS detection kit (dichloro-dihydro-fluorescein diacetate (DCFH-DA)) and bicinchoninic acid assay (BCA) protein assay kit were purchased from Beyotime Company (Jiangsu, China). HIF-1α antibody and VEGF antibody were from Proteintech Group, Inc. (Wuhan, China). β-actin antibody and horseradish peroxidase-conjugated secondary antibody were from Santa Cruz Co. Ltd. (Santa Cruz, CA). The hypoxia marker Pimonidazolehydrochloride (Hypoxyprobe-1 plus kit) was purchased from Hypoxyprobe (Burlington, MA). Roswell Park Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin (100 U/mL) were from Ji Nuo Biotechnology Co., Ltd. (Zhejiang, China). All other chemicals were of analytical grade and were used without further purification.

Tissues were fixed in 4% paraformaldehyde for 24 hours and paraffin-embedded, sectioned, dewaxed in xylene, and rehydrated through graded alcohols. Antigen retrieval was performed in citric acid buffer (pH 6.0). Sections were blocked in 2% normal goat serum for 1 hour and stained with primary antibodies as follows: anti-CD31 antibody (1:500; Abcam) for endothelium, α-SMA antibody (1:100; Proteintech) for pericytes, and HIF-1α antibody (1:50; Proteintech) for hypoxia. The sections were then washed and incubated with rhodamine-conjugated goat anti-rat IgG (H + L) (1:50; Proteintech) or goat anti-rabbit IgG-FITC (1:200; Santa Cruz) for 40 min at RT. The level of pericyte coverage was presented as a percent of the length along CD31⁺ vessels. The ratio of hypoxia in the tumour sections was presented as HIF-1α staining area/CD31⁺ area.

Bankpeptide Co., Ltd. (Hefei, China) custom-make the peptide (DEN-TAT) (sequence, K4K2KGRKKRRQRRRPPQC). 2-(2-Pyridyldithio)ethylamine hydrochloride (HY-101794-50) was purchased from MedChemExpress (Shanghai, China). Perfluorooctanoyl chloride and Cobalt chloride (CoCl₂) were from Sigma-Aldrich (St. Louis, MO, USA). TrypLETM Express, Opti-MEM^®, and HEPES buffer were from Gibco (Waltham, MA, USA). Lipo8000™, DTT, Agarose, TBE buffer, LysoTracker Green, 100 × Hoechst 33342, Calcein AM cell activity assay kit, and Propidium iodide were obtained from Beyotime (Shanghai, China). Triton X-100 was purchased from Solarbio (Beijing, China). Matrigel^® matrix was from Corning (New York, NY, USA). Sorafenib was obtained from CSNpharm (Arlington Heights, IL, USA). Glutathione was purchased from Adamas-beta (Shanghai, China). DMOG was obtained from TCL. β-Actin antibody, HIF-1α antibody, and secondary antibody were purchased from Proteintech (Rosemont, IL, USA). siRNA-targeting VEGF (siVEGF) (anti-sense strand: 5′-GAUCUCAUCAGGGUACUCCdTdT-3′, sense strand: 5′-GGAGUACCCUGAUGAGAUCdTd-3′), siRNA-targeting HIF-1α (siHIF-1α) (sense strand: 5′-CGAUCAUGCAGCUAC UACAdT dT-3′; anti-sense strand: 5′-UGUAGUAGCUGCAUGAUCGdTdT-3′), Cyanine 5 labeled siRNA (Cy5-siRNA), and negative control scrambled siRNA (siNC) were all from Genepharma (Shanghai, China).