Alexa fluor 546

Brain cryosections or treated BV2 cells were fixed with 4% paraformaldehyde (PFA) for 20 min, followed by permeabilization using a mixture of 0.5% Triton X‐100, 10% donkey serum, and 90% PBS for 60 min at room temperature. The samples underwent overnight incubation at 4°C with the following primary antibodies: anti‐Iba‐1 (1:500, rabbit, 019–19,741, WAKO, Japan), anti‐CD16 (1:500, goat, PA5‐47230, Thermo Fisher Scientific, USA), and anti‐CD206 (1:500, rat, MA5‐16871, Thermo Fisher Scientific, USA). After being washed with PBS, the brain slices/cells were incubated with secondary antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 546 (1:500, Vector Laboratories, USA) at room temperature for 60 min. Subsequently, the samples were washed three times with PBS, and then stained with 100 nM DAPI for 15 min. Immunofluorescent staining was observed and photographed using a fluorescence microscope (Olympus Corporation).

For brain immunofluorescence staining analysis, mice were anesthetized and transcardially perfused with 4% paraformaldehyde (PFA). The brains were dissected, postfixed in 4% PFA at 4 °C for 2 h, and then sectioned into 50-μm-thick slices by a VT1000S vibratome (Leica). The brain sections were pretreated in 0.5% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) for 1 h, blocked with 10% normal donkey serum, and 0.1% Triton X-100 in PBS for 1 h, and incubated with the indicated primary antibodies at 4 °C overnight. After washing with PBS, the samples were incubated with appropriate secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 546 (1:500, Vector Laboratories (Burlingame, CA, USA)) at room temperature for 1 h. The sections were counterstained with DAPI for 10 min, followed by washing in PBS. Images were acquired by ZEISS LSM880 confocal microscopy.