Fluorescent dye conjugated secondary antibodies

We harvested the left hind knees of mice after euthanasia at 2 or 4 weeks after ACLT and fixed them with 4% formaldehyde for 48 hours. Then, the knees were immersed in 1.5 M EDTA for 1-1.5 weeks for decalcification. Following dehydration in a gradient of alcohol solutions, the knees were embedded in OCT compound for sectioning (6 μm). For safranin-O & fast green staining, sections were dewaxed in water and immersed in fast green staining solution for 6 minutes. After washing with distilled water for 1 minute, the sections were immersed in safranin O solution for 4 minutes, rinsed with distilled water for 1 minute and differentiated in glacial acetic acid for 1 minute. The sections were sealed with neutral resin after dehydration. For immunohistochemical staining, sections were dewaxed in water and repaired in boiled antigen retrieval solution for 8 minutes. After blocking with a 3% BSA/PBS solution for 1 hour at room temperature, sections were incubated with anti-endomucin (Emcn; Santa Cruz, Dallas, Texas, USA), anti-matrix metalloproteinase 13 (MMP13; Abcam, Cambridge, UK) and anti- Runt-related transcription factor 2 (RUNX2; Servicebio, Wuhan, China) antibodies, followed by fluorescent dye-conjugated secondary antibodies (Abcam, Cambridge, UK) for fluorescence imaging and HRP-conjugated secondary antibodies (Servicebio, Wuhan, China) for DAB staining.

Vascular tissue was fixed with paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 μm. Specimen sections were dewaxed and incubated with anti-CD68 (Abcam, Cambridge, MA, USA) and anti-TNF-α (Abcam, Cambridge, MA, USA) antibodies at 4°C overnight, washed with PBS and incubated with fluorescent dye-conjugated secondary antibodies (Abcam). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min. Immunofluorescence images were captured using a fluorescence microscope (Dmi8+DFC 7000T, Leica, Wetzlar, Germany).

Colonic slides were incubated with antigen recovery solution (Vector Laboratories, Inc. Burlingame, CA, USA) for 15 min, and nonspecific binding was blocked with 5% BSA. Next, the slides were incubated with anti-ZO-1 and anti-β-catenin primary antibodies (ab96587 and ab32572, Abcam, Cambridge, MA, USA) at 4°C overnight. After being washed with PBS, slides were incubated with fluorescent dye-conjugated secondary antibodies (Abcam) for 1 h and protected from light. Finally, 4,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. Sections were examined with a DM5000 B fluorescence microscope (Leica, Wetzlar, Germany).
After the colonic sections were deparaffinized, DNase-free proteinase K (20 μg/mL) was applied, and the sections were incubated at 37°C for 20 min. After washing with PBS, the TUNEL detection solution was added dropwise, and sections were incubated in a 37°C incubator for 2 h. After the stop solution was added to terminate the reaction, the sections were washed with PBS. Thereafter, the streptavidin-HRP working solution was added dropwise, and sections were incubated for 30 min. The sections were washed with PBS, then treated with 0.2–0.5 mL of DAB and incubated at room temperature. The specific time was determined according to the degree of color development, and images were acquired for analysis.