Fitc conjugated goat anti rabbit igg h l

The immunohistochemistry (IHC) staining of tissues was performed as previously described [33 (link)]. The primary antibody used was the anti-FPR (1:500; Cell Signaling Technology, Danvers, MA, USA). The secondary antibody used was biotin-conjugated anti-rabbit IgG (1:200; Servicebio, Wuhan, China). The staining was visualized using diaminobenzidine (DAB; Servicebio, Wuhan, China), with brown cells indicating a positive result.
The immunofluorescence staining of the OFs was performed as previously described [34 (link)]. The primary antibodies used were the FPR (1:500; Cell Signaling Technology, Danvers, MA, USA), vimentin (1:500; Abclonal, Wuhan, China), cytokeratin (1:500; Abclonal, Wuhan, China), desmin (1:500; Abclonal, Wuhan, China), S-100 (1:500; Abclonal, Wuhan, China), and myosin (1:500; Abclonal, Wuhan, China). The secondary antibody was FITC-conjugated goat anti-rabbit IgG (H + L) (1:200; Servicebio, Wuhan, China).

After dewaxing, paraffin sections were treated with citrate buffer (pH = 6) to retrieve antigens. Then, sections were rinsed with phosphate buffer saline (PBS; Beyotime, Shanghai, China) thrice and blocked with goat serum (SP9002; ZSGB-BIO, Beijing, China) for 20 min at room temperature. Next, the sections were incubated with the iNOS antibody (1 : 100, ab178945; Abcam, Cambridge, UK), CD206 antibody (1 : 100, 24595; Cell Signaling Technology, Inc., Danvers, MA, USA), HLA-DR antibody (1 : 100, MA5-32232; Thermo Fisher Scientific, Waltham, MA, USA), CD11c antibody (1 : 100, A1508; ABclonal, Wuhan, China), ICAM-1 antibody (1 : 100, 10020-1-AP; Protein Tech, Wuhan, China), and VCAM-1 antibody (1 : 100, ab134047; Abcam) overnight at 4°C. Subsequently, the sections were washed with PBS three times and incubated with FITC-conjugated Goat Anti-Rabbit IgG (H + L) (1 : 100, GB22303; Servicebio, Wuhan, China) or Cy3-conjugated Goat Anti-Rabbit IgG (H + L) (1 : 100, GB21303; Servicebio) at 37°C for 30 min. The nuclei were stained with DAPI (ZLI-9557; ZSGB-BIO) for 10 min at room temperature. Images were captured using a confocal microscope (LSM700; Zeiss, Oberkochen, Germany).

LPS (Escherichia coli 055: B5) was obtained from Sigma-Aldrich (St. Louis, MO, USA). HMW-HA (0.8–1.5 × 10⁶ Da, 1.8 × 10⁶ Da) was obtain from Meilunbio (Dalian, China). CCK-8 and One Step TUNEL Apoptosis Assay Kit were purchased from Beyotime (Shanghai, China). DAPI solution and FITC conjugated Goat Anti-Rabbit IgG (H + L) was obtained from Servicebio (Wuhan, China). All primary antibodies used in this study were from Proteintech Group (Chicago, IL, USA) except the anti-β-actin (Medical Discovery Leader (MDL) Biotechnology, Beijing, China), anti-IL-1β (Abcam, Cambridge, UK) and anti-COL2A1 (Santa Cruz Biotechnology, Dallas, TX, USA). Bafilomycin A1 (Baf-A1) was purchased from Selleck (Houston, TX, USA). The reagents used for synthesis of o-HA derivatives were of analytical grade.