Methoxy xo 4

Mice were injected intraperitoneally with methoxy‐X‐O4 (Tocris) (10 mg/kg bodyweight), a fluorescent congo red derivate, in a DMSO/PBS mixture as described previously (Mezo et al, 2020 (link)). After 3 h, hippocampi were collected and pvMΦ were assessed as CD11b⁺CD45^hi cells. Percentage of methoxy‐XO‐4‐positive pvMΦ were determined by flow cytometry using a FACS Canto II (BD Bioscience) and analyzed with FlowJo software (Tree Star).

Mice were injected intraperitoneally with methoxy‐XO4 (Tocris) at 10 mg/kg bodyweight in a DMSO/PBS mixture at 1:10 ratio as described previously with slight modifications (Heneka et al, 2013). Hippocampi were isolated 3 h after methoxy‐XO4 injection and processed into single‐cell suspension with a potter. The homogenate was filtered through a cell strainer (70 μm) and was separated by 37% Percoll gradient centrifugation at 800 g for 30 min at 4°C. The myeloid containing phase was collected and washed once with PBS. Fc receptor blocking antibody CD16/CD32 (1:200, clone 2.4G2, BD Bioscience) was applied in order to prevent unspecific binding, and dead cells were stained using the Fixable Viability Dye eFluor^® 780 (1:1,000, eBioscience) at 4°C for 20 min. Cells were washed once and then stained with primary antibodies directed against CD11b (1:200, clone M1/70, eBioscience), CD45 (1:200, clone 30‐F11, eBioscience), and CD36 (1:200, clone 72‐1, eBioscience) at 4°C for 20 min. Cells were washed again, and then, frequencies of viable methoxy‐XO4⁺ CD11b⁺CD45^low microglia cells were determined by flow cytometry using a FACS Canto II (BD Biosciences) and analyzed using FlowJo (Tree Star). WT mice injected with methoxy‐XO4 were used as controls to determine the methoxy‐XO4 threshold for non‐phagocytosing cells. Corresponding isotype control antibodies were used.