Fluorescein fitc conjugated affinipure goat anti rabbit igg h l

The cell slides were placed in a six-well cell culture plate, and cells in the logarithmic growth phase were selected and plated on cell slides. When the cells grew to 80–90% confluence, they were fixed with 4% paraformaldehyde at room temperature for 30 min. Cells were permeabilized with PBS containing 0.5% Triton X-100 for 15 min at room temperature and washed three times with PBS solution for 5 min each. Then, incubation with the primary antibody was performed overnight at 4 °C. The next day, the cells were washed three times with PBS solution for 5 min each, and incubated with fluorescent secondary antibodies for 1.5 h at room temperature in the dark. After washing the cells three times with PBS solution, the nuclei were stained with DAPI solution for 10 min. The cells were washed three times with PBS solution, mounted on slides, and photographed under a confocal microscope (Nikon, Japan). The antibodies used were: HPV16 E6/18 E6 (C1P5) (Santa Cruz Biotechnology, sc-460); P53 Monoclonal antibody (Proteintech, 60283-2-Ig); E6AP/UBE3A Polyclonal antibody (Proteintech, 10344-1-AP); Fluorescein (FITC)–conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00003-1); Fluorescein (FITC)–conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00003-2).

Immunofluorescence staining assay was carried out as previously described.21 The primary hepatocytes were treated with adenoviral infection and fixed with 4% paraformaldehyde. The fixed cells were then treated with 0.1% Triton‐100 and blocked with 10% bovine serum albumin, followed by incubated with MLXIPL antibody (1:100‐1:200 dilution; Gen Tex; Cat#GTX30677) in 4°C overnight and stained with Fluorescein (FITC)–conjugated Affinipure Goat Anti‐Rabbit IgG (H + L) (1:20‐1:100 dilution; Proteintech, Cat#SA00003‐11). Cell nuclei were counterstained with DAPI, followed by fluorescence microscopic imaging (magnification, ×400).

Forskolin and melatonin were purchased from Sigma-Aldrich. Y-27632 and arachidonic acid (AA) were purchased from Selleck Chemicals. The following antibodies were used in this study: BMAL1 (Abcam ab3350 for immunofluorescence and Santa Cruz Biotechnology sc-365645 for western blot), aggrecan (Millipore Sigma AB1031), MMP13 (Abcam ab39012 for western blot and Proteintech 18165-1-AP for immunofluorescence), and phosphomyosin light chain 2 (Ser19; Cell Signaling Technology 3671). Horseradish peroxidase (HRP)-conjugated β-actin mouse monoclonal antibody, HRP-conjugated AffiniPure goat anti-mouse or goat anti-rabbit IgG (H + L), fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (H + L), and Cy3-conjugated AffiniPure goat anti-rabbit IgG (H + L) were purchased from Proteintech. Cell culture reagents were purchased from Gibco.