Peroxidase conjugated secondary antibody

Hearts were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin (H&E) (C0105, Beyotime Biotechnology) for histological analysis and Sirius Red and Masson’s trichrome (G1340-7 × 100 ml, Solarbio Life Science) to evaluate cardiac fibrosis. The sections were observed under a light microscope (Nikon, Japan).
For immunohistochemical staining, the sections were deparaffinized and rehydrated. The sections were treated with 3% H2O2 for 30 min to block endogenous peroxidase activity and then with 1% BSA in PBS for 30 min. The slides were incubated overnight at 4°C with the primary antibody (TNF-α, 1:50; both from Santa Cruz). Peroxidase-conjugated secondary antibodies were used for detection (Santa Cruz; 1:100 dilution; 1 h incubation). The slides were counterstained with hematoxylin for 5 min, dehydrated, and mounted. Images were viewed by a bright field microscope (Nikon).

Cells were lysed on ice for 20 min using a lysis buffer and centrifuged at 14,000 ×g for 10 min at 4°C. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were transferred onto nitrocellulose membranes and the membranes were blotted with appropriate primary antibodies at a dilution of 1:1000. The membranes were then treated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Bound antibodies were detected with Super Signal West Pico PLUS Chemiluminescent Substrate (ECL; Amersham, Arlington Heights, IL, USA) and developed with Kodak X-OMAT films (Kodak, New Haven, CT, USA). Primary antibodies used in this study were COL-1A (ab34710; Abcam, Cambridge, UK), α-SMA (ab5694; Abcam, Cambridge, UK), fibronectin (sc-69681; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), TGF-β (sc-130348; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and GAPDH (ATGEN, Seongnam-si, Gyeonggi-do, South Korea) antibodies.

Flower of Pueraria lobata Ohwi was purchased from Inno Hanbang Herbal Drug Co. Ltd. (Seoul, Korea) and a voucher specimen of the herb (PF1-2012) was deposited in the herbarium of the College of Pharmacy, Kyung Hee University. Pueraria flower (100 g) was added to 10 times of distilled water, heat-extracted (for 2 h at 100 °C), and concentrated under reduced pressure. The filtrate was lyophilised using an FDU-550R freeze-dryer (Eyela Co., Tokyo, Japan), and stored at 4 °C until use. The dried extract of Pueraria flower (PFE) was dissolved in distilled water before each experiment. Foetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium, penicillin G, and streptomycin were obtained from Life Technologies (Grand Island, NY, USA). CellTracker™ was purchased from Invitrogen (Grand Island, NY, USA). RNase, hydrocortisone, and PD98059 were obtained from Sigma Chemical Co. (St. Louis, MO, USA.). MMP-2 antibody was from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies for β-actin, MMP-9, total extracellular signal-regulated kinase (t-ERK), phospho-ERK (p-ERK), and the peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Enhanced chemiluminescence (ECL) system was purchased from Amersham Pharmacia Biotech (Oakville, ON, Canada).

GSS was purchased from the HANKOOK SHINYAK Corporation (Chungcheongnam-do, South Korea). Primary antibodies used for Western blotting were as follows: anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1 : 1000; Wako, Japan), anti-glial fibrillary acidic protein (GFAP; 1 : 3000; Millipore, MA, USA), anti-survival motor neuron (SMN; 1 : 1000; Santa Cruz Biotechnology, CA, USA), anti-TLR4 (1 : 1000; Santa Cruz Biotechnology), anti-CD14 (1 : 1000; BD Pharmingen, CA, USA), anti-COX2 (1 : 1000; Abcam, MA, USA), anti-transferrin (1 : 1000; Santa Cruz Biotechnology), anti-HO1 (1 : 1000; Abcam), anti-Bcl2 associated X (Bax, 1 : 1000; Santa Cruz Biotechnology), anti-phospho 5′-adenosine monophosphate-activated protein kinase (pAMPK; 1 : 1000; Cell Signaling, MA, USA), anti-AMPK (1 : 1000; Cell Signaling), and anti-phospho mammalian target of rapamycin (mTOR; 1 : 1000; Cell Signaling). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 : 1000; Santa Cruz Biotechnology) was used to control for protein loading. Peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

The total cellular protein of TH1 cells was extracted using RIPA lysis buffer (Thermo Fisher Scientific). Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to polyvinylidene fluoride membranes (Sigma-Aldrich). Membranes were blocked with 5% skim milk and incubated with primary antibodies against manganese-dependent superoxide dismutase (MnSOD; Santa Cruz Biotechnology, Dallas, TX, USA), p-Smad2 (Novus Biological, Centennial, CO, USA), Smad7 (R&D system, Minneapolis, MN, USA), fibronectin (Thermo Fisher Scientific), p-Akt (Santa Cruz Biotechnology), PrP^C (Santa Cruz Biotechnology), Collagen I (Santa Cruz Biotechnology), E-cadherin (Santa Cruz Biotechnology), α-smooth muscle actin (α-SMA; Santa Cruz Biotechnology), and β-actin (Santa Cruz Biotechnology). After incubation of membranes with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific) in a dark room. For quantification of band intensity, each band intensity was analyzed by ImageJ software (http://rsb.info.nih.gov/ij/). The expression levels of proteins were determined relative to the expression of β-actin.