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 incision43 (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.
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.
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