Sj1909 nbp2 67416

Paraffin sections (4 μm) of pulmonary tissues were rehydrated and microwaved in citric acid buffer to retrieve antigens. After incubation with 10% BSA for 1 h, the sections were incubated with primary antibodies against integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), fibronectin and collagen-I (72026S, CST, USA) at a dilution of 1:100 at 4 °C overnight. After washes, sections were incubated with Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA) for integrin β3, PKM2 and collagen-I, fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Invitrogen) for fibronectin at a dilution of 1:400, at 37 ℃, for 1 h in the dark. Finally, nuclei were counterstained with 4'6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA). Aipathwell softwell (Servicebio, China) was used to analyze the integrated optical density of the single or double stained fluorescent proteins for the semi-quantitative analysis.

The samples were cut into 5 mm sections and placed onto slides. Endogenous peroxidase was quenched by H2O2. Mouse monoclonal integrin β3 (SJ1909/NBP2-67416, Novus, USA) and PKM2 antibody (AF7244, R&D, USA) were added on the sections as primary antibodies. The sections were incubated with polymer for 30 min and developed in DAB solution for 5 min. Then, the sections were counterstained with hematoxylin and mounted with cover slip. The Image-Pro Plus 6.0 software was applied to select the same tan color as a uniform criterion to evaluate all positive photos, and five high-power fields of light microscope was used to obtain the cumulative optical density value.

Pulmonary tissue was homogenized with lysis buffer, resolved on 10% SDS-PAGE using a Mini-Protean 3 Electrophoresis Cell (Bio-Rad) at 150 V, 100 mA for 1 h, and transferred to polyvinylidene fluoride membrane (Millipore, HVLP04700). The membrane was washed and blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 10% nonfat dry milk before overnight incubation with appropriate primary antibodies (integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), collagen-I α-1 (COL1A1, 72026S, CST, USA), α-smooth muscle actin (α-SMA) (CST, 19245), Lactate dehydrogenase (LDHA) (3582, CST, USA) and β-actin (8457, CST, USA)) in blocking buffer at 4 °C. The secondary antibodies were then used with 1:6000 goat anti-mouse IgG-Horseradish Peroxidase (HRP) for PKM2, or 1:6000 goat anti-rabbit IgG HRP for integrin β3, COL1A1, α-SMA and β-actin, in blocking buffer for 1 hour at room temperature. The bands were visualized using enhanced chemiluminescence (Amersham ECL Western Blotting Detection Reagents, GE Healthcare, Baie d'Urfe, QC, Canada).