Nbp2 32831

Cells were seeded at 40% confluence on round glass slides in 24-well plates. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilization with 0.1% Triton X-100 (Beyotime) in PBS. After blocking in goat serum at 37 °C for 30 min, the samples were incubated with antibodies against CD68 (Novus Biologicals, USA, NBP2-32831), CD206 (NovusBiologicals, USA, MAB2534), α-SMA (Novus Biologicals, USA, NBP2-33006), or Collagen I (Novus Biologicals, USA, NB600-408) in Diluent at 4 °C overnight. The samples were then incubated with secondary antibody (CST, USA, 8889/4412) at 37 °C for 1 h, followed by washing with PBS and staining with DAPI for nuclear visualization. Cell staining was examined under an inverted fluorescent microscope. The quantification of the mean fluorescence intensity (MFI) was performed using Image-Pro-Plus version 6.0.

Serial 4-μm-thick paraffin sections from CIA synovial tissues were deparaffinized in xylene and rehydrated through a graded ethanol series. Then, the sections were immersed in the 0.01 M citrate buffer (pH 6.0), and microwave irradiation was performed three times (8 min/time) for antigen retrieval. The sections were incubated in 5% goat serum for 1 h before immunostaining. In double-labeling experiments, the sections were incubated with anti-CD68 (pAb, 1:200, ab125212, Abcam, British; mAb, 1:100, NBP2-32831, NOVUS, USA), anti-Vimentin mAb (1:200, ab92547, Abcam, British; 1 : 200, ab20346, Abcam, British), anti-TLR2 pAb (1:150, 17236-1-AP, Proteintech, China), and anti-TLR4 mAb (1 : 100, sc-293072, Santa Cruz, USA) at 4°C overnight, followed by incubated with secondary antibodies, goat anti-rabbit IgG H&L(FITC) (1 : 200, ab6717, Abcam, British) or goat anti-mouse IgG H&L (Cy3) (1:200, ab97035, Abcam, British). The poststaining sections were examined with a full-spectrum scanning confocal microscope (FV1200, Olympus, Japan), and pairs of images were superimposed for colocalization analysis.