Mice were deeply anesthetized and perfused as previously described [11 (link)]. The brain was dissected, post-fixed in 4% PFA overnight, stored in PBS with 0.01% sodium azide, and sectioned at 50 μm using Leica vibratome (Leica VT1000S, Leica Inc., Nussloch, Germany). Floating sections were processed for immunocytochemistry [10 (link)]. All antibodies were obtained from Abcam (Cambridge, MA, USA) unless otherwise stated. In this study, chicken anti-GFP (1:500), rat anti-GFAP (1:1000; Invitrogen), rabbit anti-Iba1 (1:500; Wako), TMEM 119 (1:500), mouse anti-CCR2 (1:500), and rabbit anti-CD31 (1:500) were used.
For postmortem human samples, slides were baked for 60 min at 60 °C, deparaffinized with xylene for 5 min, and rehydrated in ethanol (100, 95, 70, and 50%). Antigen retrieval was performed as previously reported [17 (link)]. In this study, chicken anti-Map 2 (1:200, Abcam, Cambridge, MA, USA), rat anti-GFAP (1:1000; Invitrogen, Thermo Fisher Scientific, Rockford, IL, USA), rabbit anti-Iba1 (1:500; Wako), TMEM 119 (1:1000), mouse anti-CCR2 (1:500), and rabbit anti-CD31 (1:500) were used.
For postmortem human samples, slides were baked for 60 min at 60 °C, deparaffinized with xylene for 5 min, and rehydrated in ethanol (100, 95, 70, and 50%). Antigen retrieval was performed as previously reported [17 (link)]. In this study, chicken anti-Map 2 (1:200, Abcam, Cambridge, MA, USA), rat anti-GFAP (1:1000; Invitrogen, Thermo Fisher Scientific, Rockford, IL, USA), rabbit anti-Iba1 (1:500; Wako), TMEM 119 (1:1000), mouse anti-CCR2 (1:500), and rabbit anti-CD31 (1:500) were used.