Horseradishperoxidase hrp labelled secondary antibody

In this experimental study, chondrocytes purchased from Yunmi Biotechnology Company
(Shanghai, China) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand
Island, NY, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, Grand Island, NY,
USA), 100 U/ml penicillin (Gibco, Grand Island, NY, USA) and 100 μg/ml streptomycin
(Gibco, Grand Island, NY, USA). The chondrocytes were incubated at 37˚C under humid
conditions and 5% CO₂ . Once the chondrocytes reached 80-90% confluency, they
were passaged.
An inverted microscope (Olympus IX70) was utilised
to observe the morphology of the chondrocytes. The
chondrocytes were identified by type II collagen
immunohistochemistry staining. Briefly, chondrocytes
were incubated overnight with anti-collagen II antibody
(1:100; 15943-1-AP, Proteintech, Chicago, IL, USA).
Thereafter, the chondrocytes were treated with horseradish
peroxidase (HRP)-labelled secondary antibody (Jackson
ImmunoResearch Laboratories, Inc., USA) at 37˚C for
one hour. The colour was developed by diaminobenzidine
(DAB, Beyotime Biotechnology, China), and the cells were
counterstained with hematoxylin (Servicebio, China).
This research was performed after receiving the
ethics approval from the Ethics Communication of First
Affiliated Hospital of Zhejiang University (2018-IIT-34).

The total cellular proteins were extracted from treated keratinocytes with lysis buffer (1.5% SDS, 0.0625 M Tris–HCl and 1 mM Na₃VO_4, pH 6.8) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). For Western blotting analysis, 40 μg of extracted total cellular protein was subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. After blocking and washing, the membrane was incubated with antibodies including phosphorylated extracellular signal-regulated kinases (pERK), total-ERK (tERK), and GAPDH (Cell Signaling, Beverly, MA), followed by incubation with horseradish peroxidase (HRP)-labelled secondary antibody (1:5000; Jackson Immuno Research, West Baltimore Pike, PA) and developed using a commercial HRP substrate (Immobilon™ Western Chemiluminescent HRP Substrate; Millipore Corporation, Billerica, MA). The membranes were detected by ChemiDoc™ XRS (Bio-Rad Laboratories Inc., Hercules, CA).