Ain 76a standard chow

PLX3397 was provided by Plexxikon and formulated in AIN-76A standard chow by Research Diets (290 p.p.m.; 290 mg PLX3397 in 1 kg chow). Mice were fed a PLX3397 diet for 3 weeks to eliminate microglia24 (link). One group of mice was given PLX3397 for 3 weeks, followed by withdrawal of the drug by replacing the PLX3397 diet with control chow diet for 24 h before experiments. Another group of mice was fed PLX3397 for 3 weeks followed by 2 weeks on control diet to allow for the repopulation of microglia in the brain. In agreement with earlier results24 (link), we have not found any obvious behavioural alterations, weight loss or sign of illness in mice fed a PLX3397 diet for 3 weeks or longer. Individual mice were observed for a 5-min period once daily for any changes in activity, exploratory behaviour and social interaction. Baseline temperature was recorded for every mouse before surgery or imaging. No mice were excluded from these studies due to fever, weight loss, infection or behavioural alterations as a result of PLX3397 diet.

C57BL/6J mice of both sexes were used for scRNA-seq at 11–14 weeks or 18–22 months of age. PDMCAO was induced by permanent ligation of the right distal middle cerebral artery (MCA) using a micro-coagulator (Accu-temp)^{11 (link)}. Mice were anesthetized with isoflurane (4% induction and 2% maintenance in airflow) and body temperature was maintained at 37°C by feedback-controlled heating pad and rectal temperature probe. Bupivacaine (0.25% at 1ml/kg) was injected subcutaneously (s.c.), prior to any skin incision^{43 (link)}. The distal MCA was accessed via a craniotomy and permanently occluded just proximal to the anterior and posterior branches by electrocoagulation. Sham controls were generated with same procedure without electro-coagulation of the MCA.
For microglia depletion experiments, we used PLX5622, a CSF1R antagonist (REF). PLX5622 was provided by Plexxikon Inc. (Berkeley, CA) and formulated in AIN-76A standard chow at 1200 ppm by Research Diets Inc. PLX5622 was administrated for 7 days prior to the PDMCAO procedure and continued for 3 days after stroke. At 3 days after surgery, brains were isolated and ipsilateral hemisphere was used for RNA isolation and qRT-PCR analysis. The contralateral hemisphere was used for immunostaining.

Animals received subcutaneous injections of BLZ (MW: 398.48, MedChem Express HY-12768/CS-3971), a small molecule inhibitor of CSF1R^{16 (link)}, dissolved in dimethylsulfoxide (DMSO; D136-1; Fisher Scientific) to eliminate microglia as previously described^{9 (link)}. Injections of 0.01 ml of BLZ solution (200 mg/kg) or control (DMSO) were administered subcutaneously every 2 or 3 days, depending on treatment group, starting from P2 with the last injection delivered at P12 or P15. Groups receiving an additional CSF1R inhibitor were fed a diet containing PLX5622 (PLX) or the respective control rodent diet ad libitum (provided by Plexxikon Inc. and formulated in AIN-76A standard chow by Research Diets Inc, 1200 mg/kg chow)¹⁷ . Litters were given PLX or the respective control (CTL) through the milk of the lactating mother. The mother was given PLX or CTL when the litter was P2, the same day that BLZ945 or DMSO injections were administered to pups. The mother remained on the chow until the litter was of age to wean (P21), and pups had access to the PLX or control chow when they began a solid diet (~ P16). After weaning, BLZ/PLX treated mice remained on the PLX chow until testing at either 4 or 7 weeks of age.

PLX3397 was provided by Plexxikon Inc (Berkeley, CA) and formulated in AIN-76A standard chow by Research Diets Inc at the doses indicated in the text. PLX3397 was provided in chow at 600 ppm.

Microglia depletion and subsequent repopulation was achieved through administration of the colony stimulating factor 1 receptor (CSF1R) inhibitors PLX5622 (1200 ppm delivering daily doses of 168 mg/kg) (Plexxikon, Inc., mixed into AIN-76A standard chow by Research Diets, Inc.) from postnatal day P21-42 (MG-REP group) [14 (link), 15 (link)].

Control and PLX5622 (300 ppm formulated in AIN-76A standard chow, Research Diets, Inc.) chows were provided by Plexxikon Inc (Berkeley, CA). Approximately 1.2 mg of PLX5622 was ingested by each mouse per day (calculation based on 4 g/mouse chow daily).