Fitc conjugated anti foxp3

Synovia from mice knees were fixed in 4% paraformaldehyde (PFD) for 48 h, decalcified in 10% EDTA for 1 week, and then kept in 30% sucrose until they were cut into 7 μm thick sections. Antigen retrieval was performed by microwave treatment for 10 min in 10 mM citrate buffer solution (pH 6.0). Sections were deparaffinized, rehydrated, and treated with 10% goat blocking serum for 20 min and incubated with anti-TNF-α (Abcam, Cambridge, MA, USA) overnight at 4 °C. After rinsing with PBS for 15 min, tissues were incubated with goat anti-rabbit Alexa Fluor 568 (BD Biosciences) at room temperature. Spleens from mice were collected, embedded in Tissue-Tek Optimal Cutting Temperature compound (Sakura Finetek, Nagano, Japan), and snap-frozen in liquid nitrogen. Cryosections (7 μm thick) were fixed with 4% PFD, blocked with 10% goat serum for 20 min, and stained using FITC-conjugated anti-Foxp3 and APC-conjugated anti-CD25 (all from eBioscience) overnight at 4 °C. Nuclei were counterstained with 2 μM 4′, 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) for 5 min. Thereafter, sections were washed twice in PBS for 10 min and twice in distilled water for 10 min, mounted in ProLong Gold anti-fade reagent (Cell Signaling Technology, Boston, MA, USA), and imaged using an inverted fluorescence microscope (EVOS FL Cell Imaging System; Life Technologies, Darmstadt, Germany).

The number of circulating EPCs and Tregs were detected by flow cytometry. The details of flow cytometry were described in our previous paper [20 (link)]. For analysis of EPC cell surface molecules, cells were stained with the following antibodies: unconjugated rabbit anti-CD133 (Abcam, Cambridge, UK), PE-conjugated anti-CD34 (Santa Cruz Biotech, Santa Cruz, CA), VEGFR2⁺ (BD Pharmingen, San Diego, CA) and goat anti-rabbit FITC secondary antibody (Abcam, Cambridge, UK). For analysis of Treg cells, cells were stained with PE-Cy5-conjugated anti CD4 (eBioscience, San Diego, CA) and PE-conjugated anti CD25 (BD Pharmingen, San Diego, CA), and then permeabilized with fixation/permeabiliation working solution and incubated with FITC-conjugated anti-Foxp3 (eBioscience, San Diego, CA).