Synaptophysin antibody

FM1‐43, tetramethylrhodamine‐dextran (TMR‐dextran), penicillin/streptomycin, phosphate‐buffered salts, and Minimal Essential Medium were obtained from Invitrogen. Foetal calf serum was from Biosera. The dynamin I phospho‐specific Ser‐774 antibody was from AbD Serotec, the synaptophysin antibody was from Synaptic Systems and the synaptobrevin II antibody was from Nventa Biopharmaceuticals. Glutaraldehyde and osmium tetroxide were from Agar Scientific. Tetanus toxin (TnTx), BAPTA‐AM (1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid) and EGTA‐AM were from Calbiochem. Fura‐2 was from Anaspec. Synaptophysin‐pHluorin (sypHy) was a gift from Prof. Leon Lagnado (University of Sussex). All other reagents were from Sigma.

We used the amount of microglia-phagocytosing synaptosomes and TRITC-Dextran 40 kDa (AS026, Abclonal) (Xue et al., 2022 (link)) to assess the changes in the phagocytosis of microglia post CX3CL1 and hypoxia treatment. The synaptosome mixture was centrifuged at 150,000 × g for 30 min and the precipitate was resuspended in cell medium. Thereafter, primary microglia were separately incubated with 100 μg/mL TRITC-Dextran 40 kDa or synaptosome mixture for 30 min. The cells were then fixed and stained with a synaptosomal marker Synaptophysin antibody (101011, Synaptic Systems) using immunofluorescence. Images were recorded by a Leica SP8 confocal microscope to quantify the fluorescence intensity of synaptosomes or TRITC-Dextran in the cells.