Alexa fluor 594 conjugated anti rabbit secondary antibody

HuR-silenced and control NP cells were plated on glass coverslips and cultured in hypoxia for 72 h. After treatment, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed with PBS and then blocked with 5% normal goat serum in PBS with 0.3% Triton X-100 (Sigma Aldrich, T8787) for 1 h at room temperature. Cells were then incubated with anti-Collagen I antibody (1:500, Abcam, ab34710), anti-Collagen II antibody (1:100, Fitzgerald, 70R-CR008), or anti-Aggrecan antibody (1:100, EMD Millipore, AB1031) in blocking buffer at 4°C overnight. Cells were washed with PBS and incubated with Alexa-fluor-594-conjugated anti-rabbit secondary antibody (1:800, Jackson ImmunoResearch Lab, 711–586-152) for 1 h at room temperature. After washing with PBS, cells were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, P36934) and visualized using a Zeiss AxioImager A2 (Carl Zeiss, Germany).