Rabbit anti mouse secondary antibody

Glucose-Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Trizol reagent, miRNA reverse transcription kit, and Lipofectamine 2000 were purchased from Life Technologies (Waltham, MA). BCA protein assay kits were purchased from Beyotime (Haimen, China). Pre-miR-33a-5p lentivirus plasmid, anti-miR-5p lentivirus plasmid, HIF-1α overexpressing plasmid, and HIF-1α shRNA plasmid were purchased from DingGuoChangShengBiotech (Beijing, China). Sequences of the insert in plasmids were listed in Supplementary Table 1. miRNA qRT-PCR Detection Kit was purchased from GeneCopoeia, Rockville, MD, USA. RevertAid^™ H Minus First Strand cDNA Synthesis Kit was purchased from Fermentas. MTT was purchased from Biosharp (Hefei, Anhui, China). The Power SYBR Green kit was purchased from TOYOBO (Osaka, Japan). The glucose assay kit, lactate assay kit, and LDH activity assay kit were from Biovision (Milpitas, CA). The ATP assay Kit was from Beyotime Biotechnology (Shanghai). Mouse anti-HIF-1α monoclonal antibody, mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody, and rabbit anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK) and anti-lactate dehydrogenase A (LDH-A) antibody was from Epitomics (Burlingame, CA).

The OVA antigen was purchased from Sigma (A8040, USA) and aluminum hydroxide as adjuvant was from Kelong chemical factory (lot number 201110328, Chengdu, Sichuan, China). Enzyme-linked immunosorbent assay (ELISA) kits, specific for IgE (sIgE), interleukin 4 (IL-4), interferon-γ (IFN-γ), TNF-α, and IL-6 were purchased from Abcam (England). Antibodies against SP, GFAP, and CD11b (OX42) and rabbit anti-mouse secondary antibody were also purchased from Abcam, England. Where mentioned, biotin-conjugated rat anti-mouse antibodies were purchased from BD Pharmingen, Beijing, China. Sodium citrate buffer (0.01 M, pH 6.0) was prepared for dilution, where needed. The microscopic image acquisition and analysis system were, respectively, from BA200 Digital and Image-Pro Plus 6.0 (Media Cybernetics, USA).

Western blot assay was performed to determine the protein expression levels in each group. The cells were lysed in cold radioimmunoprecipitation assay buffer (Invitrogen Life Technologies, Carlsbad, CA, USA). A BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentrations, according to the manufacturer’s instruction. Subsequently, the proteins were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% fat dry milk in PBS for 4 h. Subsequently, the PVDF membrane was incubated with specific primary antibodies (mouse anti-cyclin D1, mouse anti-PCNA, mouse anti-CDK4, mouse anti-smooth muscle-α-actin, mouse anti-smoothelin, mouse anti-desmin, mouse anti-phospho-Akt, mouse-anti-Akt, anti-phospho-ERK, mouse-anti-ERK, and mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibodies; all from Abcam, Cambridge, UK) for 3 h. After washing three times with PBS (5 min each time), the PVDF membrane was incubated with a rabbit anti-mouse secondary antibody (Abcam). Next, after washing three times with PBS (5 min each time), an enhanced chemiluminescence western blotting kit (Thermo Fisher Scientific) was used to detect the immune complexes present on the PVDF membrane.

All the sections were blocked with 1% goat serum albumin for 1 h and then incubated with mouse monoclonal anti-CD31 primary antibody (Ab955, 1:200; Abcam) at 4 °C overnight. The sections were then stained with rabbit anti-mouse secondary antibody (1:1000; Abcam) for 1 h at room temperature. The tissue slices were washed and mounted with medium containing DAPI. All slices were observed by the Olympus FV1000 laser confocal microscope.

Cells were lysed in cold RIPA buffer (Life Technologies). The BCA Protein Assay Kit (Life Technologies) was used to determine the protein concentration. Protein was then separated with 10% SDS-PAGE, and transferred to a PVDF membrane. The PVDF membrane was blocked in 5% nonfat dried milk in PBS for 4 h. After that, the PVDF membrane was incubated with the following primary antibodies for 3 h: rabbit polyclonal anti-COL1A2 (cat no. ab208638; 1:400), mouse monoclonal anti-VEGF-A (cat no. ab155944; 1:100), rabbit polyclonal anti-Notch2 (cat no. ab137665; 1:500), rabbit monoclonal anti-Jagged2 (cat no. ab109627; 1:5000), rabbit monoclonal anti-MMP7 (cat no. ab205525; 1:4000), mouse monoclonal anti-GAPDH antibody (cat no. ab181602; 1:200). All the antibodies were purchased from Abcam, Cambridge, UK. After washing with PBS three times for 5 min, the PVDF membrane was incubated with the rabbit anti-mouse secondary antibody (1:20,000; all antibodies were purchased from Abcam, Cambridge, UK). After washing with PBS three times for 5 min, an ECL Western Blotting Kit (Millipore, Darmstadt, Germany) was used to detect the immune complexes on PVDF membrane.