Preparation of clodronate liposomes:

The production of clodronate liposomes was modified from previous descriptions (Van Rooijen, 1989; Zelinka et al., 2012). Briefly, 8 mg of L-α-Phosphatidyl-DL-glycerol sodium salt (Sigma P8318) was dissolved in chloroform. 50 mg of cholesterol dissolved in chloroform was added to the lipids and evaporated under nitrogen/vacuum with frequent shaking to create a thin lipid-film on a round bottom flask. 158 mg dichloro-methylene diphosphonate (clodronate; Sigma-Aldrich) in sterile PBS was added and mixed. To facilitate lipid rehydration, the vial was vortexed for 5 minutes. To normalize lipid vesicle size, the mixture was sonicated at 42,000 Hz for 6 minutes. The liposomes were centrifuged at 10,000 × g for 15 minutes, aspirated, and resuspended in 150 µl PBS. While there is some loss of liposomes during the purification, the dosage has been tittered such that >99% of the microglia are ablated 2 days after administration.