In situ pla probe anti mouse minus

B cells were isolated from wild-type and Cd22^−/− splenocytes using negative selection kits. In some experiments, B cells were pre-treated with Arthrobacter Uereafaciens sialidase (Roche) at 125 mU/mL to remove α2-6 Sia modifications. Vehicle control treated (PBS) or sialidase-treated purified B cells were cytospun on to a 1 cm² area of a slide at a concentration of 1.5 × 10⁵/100 μL for 7 min at 700 rpm in the Cyto-Tek Centrifuge (Model No. 4324). The cell sections were fixed using 4% PFA for 5 min at 4°C, followed by washing with PBS three times. All subsequent incubations were carried out in a humidity chamber at 37°C following the Duolink Proximity Ligation assay kit (Sigma) instructions. The following combination of primary antibodies used were: mouse anti-mouse CD22 (Cy34.1) and goat anti-mouse β₇ or goat anti-mouse β₁; and goat anti-mouse β₁ and rat anti-mouse α₄ (PS/2) followed by In Situ PLA Probe Anti-Mouse MINUS and Anti-Goat PLUS (Sigma). At the end of the PLA procedure, the sections were stained with DAPI for the identification of nuclei. Images were captured on a Zeiss LSM 880 confocal microscope using the Zeiss Zen software, a 63× oil-immersion objective and 1.5× digital zoom. The number of PLA spots per image were quantified using Imaris (v.2.0.0-rc-49/1.51d).