Spa 895 f

Immunohistochemistry was conducted using the avidin-biotin technique as described previously^{17 (link), 18 (link)}. The primary antibodies were polyclonal rat anti-macrophages/ monocytes (MOMA-2, Abcam, ab33451, 1:200), polyclonal rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, ab15323, 1:200), polyclonal rabbit anti-YM-1 (Abcam, ab93034, 1:200), monoclonal rabbit anti-ferritin (Abcam, ab75973, 1:200), and polyclonal rabbit anti-heme oxygenase-1 (HO-1, Enzo, SPA-895-F, 1:500). Negative controls omitted the primary antibody. In this study, we also use hematoxylin counterstaining for the nucleus detection. Briefly, the sections used for immunohistochemistry was immersed into hematoxylin for 10 seconds right after the 3,3’-diaminobenzidine (DAB) incubation. An oil immersion lens was adopted for morphological observations.

HO-1 (Enzo, SPA-895-F, 1:500) and donkey anti-rabbit IgG (H + L) Alexa Fluor 488 (1:500; Invitrogen) were the primary and secondary antibodies for immunofluorescent labeling. Sections were blocked with 15% donkey serum for 30 minutes and then incubated at 4°C overnight with the primary antibody. They were then incubated with secondary antibody for 2 hours. Nucleus staining was performed with 4’, 6-diamidino-2-phenylindole (DAPI) before mounting.

Immunohistochemistry was performed using the avidin-biotin-peroxidase complex techniques as previously reported^{9 (link)}. Mice were overdosed with sodium pentobarbital and underwent transcardiac perfusion with 4% paraformaldehyde (PFA). Subsequently, brains were harvested, kept in 4% PFA overnight, and immersed in 30% sucrose at 4°C. OCT compound embedded brains were sliced into 18 μm sections on a cryostat microtome, dried and preserved at −80°C. The primary antibodies were polyclonal rabbit anti-DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa, Cell Signaling, 1:400), monoclonal rat anti-MOMA-2 (monocyte + macrophage antibody, Abcam, ab33451, 1:200), polyclonal rabbit anti-HO-1(heme oxygenase-1, Enzo, SPA-895-F, 1:500). Negative controls were executed by eliminating primary antibody.