Peroxidase conjugated affinipure goat anti rabbit igg

Immunoblotting analysis was performed as described previously (Kamat et al., 2013 (link)). Briefly, approximate 100 mg frozen lung tissue powder (n = 5 in each group) was lysed in 1 mL RIPA buffer containing 100 mM PMSF to extract pulmonary total proteins. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Equal amount of proteins (10 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane (BioRad). The blots were blocked with 5% (w/v) skimmed milk in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween- 20, pH 7.5) for 2 h. Then blots were incubated with primary antibodies overnight at 4°C. Subsequently blots were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 37°C. After ECL substrates were added, the blots were analyzed using light imaging system (Tanon 5200, China). Before each step, the blots were washed five times for 5 min with TBST.
In this study, β-actin (Proteintech) was determined as an internal control of the western blot. The primary antibodies were activator of transcription 1 (STAT1) (Beyotime Biotech, China), STAT3 (Wanleibio, China), p-STAT1 Y701 (Cell Signaling Technology) and p-STAT3 Y705 (Cell Signaling Technology). All secondary antibodies were peroxidase-conjugated affinipure goat anti-rabbit IgG (Proteintech).

Human NP cells were seeded into 24-well plates and allowed to expand to near confluence. At this point, cells were incubated with CoPP (10 μM) in the presence or absence of IL-1β (10 ng/mL) for 15 days, with replacement of medium and treatment every 4 days. Cells were fixed with 4% formaldehyde in PBS for 30 min at 4 °C and rabbit anti-collagen type II polyclonal antibody (1:100 dilution) (Proteintech) was used to examine the expression of collagen type II. Peroxidase-conjugated affinipure goat anti-rabbit IgG (1:1000 dilution) (Proteintech) was used as the secondary antibody.