Horseradish peroxidase

A monoclonal antibody against human hypoxia-inducible factor-1α (HIF-1α; 1:200; Proteintech, Wuhan, China) and a monoclonal antibody against rabbit matrix metalloproteinase-9 (MMP-9;1:500; Servicebio, Wuhan, China) were performed as the primary antibodies in the present study, respectively. As for secondary antibodies, the horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G antibody (1:200; Servicebio, Wuhan, China) were used.

All tissue samples were embedded in paraffin and cut into 3-μm slices. Next, they were dewaxed with serial 75% and 85% alcohol solutions, followed by washes with absolute ethyl alcohol and Xylene I/II/III solutions. The samples were then placed in autoclaved citric acid buffer (pH 6.0) and boiled for 15 min, and then on 3% hydrogen peroxide and incubated for 20 min. The tissue slices were then coated overnight at 4 °C with anti-FGL1 polyclonal antibody (1:100 dilution; Proteintech, Rosemont, IL, USA). The samples were washed three times times with phosphate buffer saline. Next, a secondary antibody conjugated with horseradish peroxidase (1:200 dilution; Servicebio, Wuhan, China) was incubated at 25 °C for 50 min. The samples were washed three times and 3,3-diaminobenzidine (Servicebio) was used to detect positive antibody binding. The samples were counterstained with hematoxylin.

HUVECs were collected after 24 h of ox-LDL (100 µg/ml) stimulation alone or along with 12 h of PCB2 (1 µg/ml) treatment and then lysed using the RIPA lysis buffer (Beyotime Biotechnology) supplemented with protease inhibitor cocktail (Beyotime Biotechnology). After the quantitative analysis using the BCA Protein Assay Kit (Beyotime Biotechnology), protein (35 µg per lane) was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Next, the membranes were sequentially incubated with 5% skimmed milk (1 h, room temperature), the primary antibody against LOX-1 (cat. no. 17958-1-AP, Proteintech, Wuhan, China), nuclear factor kappa-B (NF-κB) p65 (cat. no. 80979-1-RR, Proteintech, Rosemont, IL, USA), IKBα (cat. no. 10268-1-AP, Proteintech), IKBα (phospho S36) (cat. no. ab133462, Abcam, Cambridge, UK), Lamin B (cat. no. 12987-1-AP, Proteintech), GAPDH (cat. no. GB11002, Servicebio, Wuhan, China), or β-actin (cat. no. GB11001, Servicebio) and a secondary antibody conjugated with horseradish peroxidase (1 h, room temperature) (cat. no. G1213, Servicebio, Wuhan, China) were used. Finally, Pierce ECL Western Blotting Substrate (Thermo Scientific) was used to detect the protein signals.