Dm6000 cfs

Cells were fixed with 4% formaldehyde and pre-incubated with 10% normal goat serum (710,027, KPL, USA), and then incubated with primary antibodies anti-Bach2 (ab83364, Abcam) and anti-HO-1 (ab13248, Abcam). Secondary antibodies were fluoresce-labeled antibody to rabbit IgG (021516, KPL, USA) and rhodamine-labeled antibody to mouse IgG (031806, KPL, USA). DAPI (157,574, MB biomedical) was used for nuclear counter staining. Images were captured by confocal microscopy (DM6000 CFS, Leica) and processed by LAS AF software.

Immunofluorescence staining was performed on 4 μm paraffin cross-sections from the carotid arteries. VSMCs were prepared as described above. The sections were deparaffinized with xylene and rehydrated, cells were fixed by 4% paraformaldehyde and then were permeabilized by incubation with 0.1% Triton X-100 in PBS. Non-specific sites were blocked by incubation in 10% normal goat serum (710027, KPL, USA) for 1 h. Then the sections or cells were incubated with primary antibodies at 4 °C overnight. The primary antibodies were anti-SMα-actin (mouse monoclonal 1:100, sc-130617, Santa Cruz), VCAM-1 (rabbit monoclonal, 1:100, ab134047, Abcam), anti-OTUD7B (rabbit polyclonal, 1:50, 16605-1-AP, Proteintech), anti-NMHC IIA (rabbit polyclonal, 1:50, 11128-1-AP, Proteintech), anti-NMHC IIA (mouse monoclonal, 1:50, 60233-1-Ig, Proteintech). Secondary antibodies were rhodamine-labeled antibody to rabbit IgG (1:50, 031506, KPL, USA) and fluorescein-labeled antibody to mouse IgG (1:50, 021815, KPL, USA), or rhodamine-labeled antibody to mouse IgG (1:50, 031806, KPL, USA) and fluorescein-labeled antibody to rabbit IgG (1:50, 021516, KPL, USA). Nuclei were stained with DAPI (1:50, 157574, MB biomedical) in each experiment. Images were captured by confocal microscopy (DM6000 CFS, Leica) and processed by LAS AF software.

The cells were fixed with 4% formaldehyde for 15 min and washed with PBS. Then, the cell smears were preincubated with 10% normal goat serum (710027, KPL) for 30 min. The cell smears were incubated with the primary antibodies anti‐ROR2, anti‐SMS1, anti‐FOXO1, and anti‐P21 at 37°C for 2 h. The fluorescence‐labeled rabbit IgG antibody (021516, KPL) was used as the secondary antibody. Finally, the cell smears were incubated in DAPI for 15 min for nuclear counterstaining. Images were acquired using confocal microscopy (DM6000CFS, Leica) and digitized with LAS AF software.

Immunofluorescence staining was performed with 5 μm paraffincross-sections from the human cervical tissues. After deparaffinized in xylene and rehydrated in graded alcohol, the slides were pre-incubated with 10% normal goat serum and then incubated with primary antibodies rabbit anti-KLF5 (1:50, GTX103289, GeneTex), mouse anti-TNFRSF11a (1:50, MBS245189, Mybiosource). Secondary antibodies were fluoresce in labeled antibody to mouse IgG (021815, KPL, USA), rhodamine labeled antibody to rabbit IgG (031506, KPL, USA). In each experiment, DAPI (0.1 ug/ml, 157574, MB biomedical) was used for nuclear counter staining. Images were captured by confocal microscopy (DM6000 CFS, Leica) and processed by LAS AF software.

Cultured human mdDCs were fixed in 4% paraformaldehyde for 5 mins at room temperature and then washed with PBS. In situ hybridization was performed on samples with specific probes for circITGB1 and miR-342-3p using a miRCURY LNA microRNA ISH Optimization Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. Fluorescence-labeled probes in a hybridization buffer were incubated with samples at 55°C for 1 h. DAPI (157574, MB Biomedicals) was used for nuclear counterstaining. Images were captured using confocal microscopy (DM6000 CFS, Leica, Switzerland) and processed with LAS AF software.

The cells were fixed with 4% paraformaldehyde for 20 minutes, then pass through the permeation and blocking procedure before being incubated overnight with the primary antibody at 4 °C. Cells were washed three times with PBS, followed by Alexa Fluor 488-conjugated secondary antibody and DAPI staining in dark condition. P65 protein and nuclei were observed by confocal fluorescence microscopy (Leica, TCS-SP5, DM6000-CFS). As for observation the nuclei translocation of NFATc1 after adding IFN-λ1, RAW264.7 cells were incubated with RANKL for 24 h in the presence or absence of 100 ng/ml IFN-λ1. Additional procedures were as same as previous descried. Gray value was elevated by using Image J software (the National Institutes of Health, USA).