HAPI microglia cells were fixed in 4% PFA for 10 min and rinsed with PBS three times. The samples were permeabilized with 0.03% Triton X-100 for 10 min, and then blocked using 1% (w/v) BSA in PBS for 30 min and incubated with primary antibodies CD40 (1:10, sc-514493, Santa Cruz Biotechnology, Santa Cruz-CA, USA) and Iba-1 (1:200, ab178846, Abcam) at 4 °C overnight. After washing off the primary antibody with PBS, secondary antibodies Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG (H+L) (1:100, 115-545-003, Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor® 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) (1:100, 711-585-152, Jackson ImmunoResearch, West Grove, PA, USA) were applied at 37 °C in the dark for 30 min. DAPI (C1002, Beyotime, Shanghai, China) was used to counterstain nuclei for 10 min in the dark at room temperature. Images were acquired at room temperature with an Olympus FV1000 spectral confocal microscope (Olympus Corporation, Tokyo, Japan) with an UPLFLN 40× objective lens (NA 1.30) and Olympus FLOWVIEW acquisition software. To quantify fluorescence, the fluorescence intensity under different transfection conditions were analyzed by Image J software (http://rsb.info.nih.gov/ij).

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