Rabbit anti eea1 antibody

Cells grown on sterile coverslips were incubated with TSG101-mCherry/CD63-EGFP-labeled EVs at 40% confluency. After 20–30 min, cells were washed extensively with PBS and then fixed with 4% paraformaldehyde for 20 min at room temperature followed by permeabilization with 0.3% Triton X-100 in PBS for 30 min. The samples were incubated with a blocking buffer containing 10% goat serum (NGS) in PBS for 2 hr at room temperature followed by addition of rabbit anti-Iba1 antibody (1:1,000; Wako Laboratory Chemicals) or rabbit anti-NF-κB p65 antibody (1:1,000; Abcam) or rabbit anti-EEA1 antibody (1:200; Cell Signaling Technology) and incubated overnight at 4°C. Primary Abs were labeled with secondary anti-rabbit Abs conjugated to the fluorescent probes Alexa Fluor 488 or Alexa Fluor 594, and nuclei were labeled with DAPI.

For BMMSC-EV uptake analysis, fixed OA chondrocytes were incubated with 2% BSA for 30 min followed by 1 h incubation with a rabbit anti-EEA1 antibody (1:200; Cell Signaling) and mouse anti-LAMP-1 (1:400, BD Pharmigen) followed by subsequent incubation with a donkey-anti-rabbit antibody labelled with DyLight 549 (Jackson) and a goat-anti-mouse antibody labelled with DyLight 649 (Jackson), respectively. For nuclear staining, DAPI was used. Images were recorded on a Zeiss LSM 700 confocal microscope (Germany). The collected z-stacks were 0.36 μm.
For NFκB p65 nuclear translocation analysis OA chondrocytes were cultured in 6-well plates and plated on glass coverslips, fixed directly after treatment in 0.1 M phosphate buffer containing 3.7% formaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min. Thereafter, cells were incubated with 2% BSA for 30 min followed by 1 h incubation with a rabbit anti-NFκB p65 antibody (1:100; Cell Signaling) and subsequent incubation with a goat-anti-rabbit-Ig antibody labelled with Alexa 681 (Molecular Probes). For nuclear staining, DAPI was used. Images were recorded on a Zeiss LSM 700 confocal microscope (Germany). The quantification of NFκB p65 positive nuclei was done using the ImageJ Plugin “Cell counter”. The NFκB p65 positive nuclei were calculated as percentage of total nuclei labelled with DAPI.