Anti f4 80 cl a3 1 alexa fluor 488

CIA was induced in DBA/1 mice. Mice were sacrificed under anesthesia and ankles exhibiting different arthritic scores were removed and fixed in 4% paraformaldehyde solution for 24 hours. Ankles were decalcified in 10% EDTA pH 7.4 (Sigma, USA) for 4 weeks (EDTA solution refreshed once a week) then embedded in paraffin. 8 μm sections were prepared from paraffin-embedded tissues. The sections were incubated in 1% blocking reagent (Roche) in PBS (1% PBS/BR) containing detection antibodies. Anti-F4/80(Cl:A3-1)/Alexa fluor 488 was purchased from AbD Serotec and anti-HA-tag (16B12)/Alexa Fluor 594 was purchased from Invitrogen. Each section was incubated overnight with 2 μg of antibody and Nb. Specific labelling was detected with anti-HA/Alexa fluor 549 in 1% PBS-BR. Fluoro-Gel II/DAPI (Electron Microscopy Sciences) mounted slides were used for fluorescence microscopy with a UPlanFI 10x, 20x or 30x objective on a OLYMPUS BX51 microscope with OLYMPUS DP71 CCD and OLYMPUS DP Controller software.

Nb119 and NbBCII10 monoclonal antibody were labelled using alexa fluor-647 lightning-link APC conjugation kit (Innova biosciences, San Diego, CA, USA). Non-activated macrophages were harvested from the peritoneal cavity of mice. About 5 × 10⁵ cells were washed three times with PBS-2% FCS and resuspended in a total volume of 100 µL. Anti-F4/80 (Cl:A3-1)/Alexa fluor 488 and Anti-Mouse CD11b (M1/70)/PE antibodies were purchased from AbD Serotec and Ebioscience. Anti-Vsig4 (NLA14) monoclonal antibody and rat IgG2a kappa isotype control were purchased from Ebioscience. Five µg of antibody or nanobody were used per 1 × 10⁶ cells for 20 min at 4 °C. Excess fluorescein labelled antibody was removed by washing with PBS-2% BSA. Stained cells were further analyzed by flow cytometry and histograms were prepared using FlowJo software (Becton Dickinson, San Jose, CA, USA).

Inflamed joints of CIA mice were fixed in 3% paraformaldehyde (pH 7.4) and then decalcified for two weeks in 10% EDTA (pH 7.4). The joints were embedded in Tissue-Tek OCT and frozen in liquid nitrogen. Cryostat sections (5 µm) were either stained with hematoxylin and eosin as standard protocol and either incubated in 1% PBS/BR containing detection antibodies for immunofluorescence microscopy. Anti-F4/80 (Cl: A3-1)/Alexa Fluor 488 was purchased from AbD Serotec and Nb was labelled by Alexa Fluor 594 (Alexa Fluor 594 NHS Ester, Shanghai, China) purchased from Invitrogen. 2 µg of antibody and Nb in 1% PBS-BR were used per slide. Fluoro-Gel II/DAPI (Electron Microscopy Sciences, Shanghai, China) mounted slides were used for fluorescence microscopy with an UPlanFI 10×, 20× or 30× objective on an OLYMPUS BX51 microscope with OLYMPUS DP71 CCD and OLYMPUS DP Controller software (Shanghai, China).