Immunohistochemical Analysis of Human Brain

Immunohistochemistry was performed as previously published [33 (link)]. In brief, human brain sections were dewaxed in xylene and rehydrated in ethanol. Antigen retrieval was performed in citrate buffer. Following blocking, samples were incubated with primary antibodies: rat anti-lipocalin-2 (1:500, R&D Systems, MAB1757), mouse anti-IBA1 (1:300, Invitrogen, MA5-27726) or rabbit anti-GFAP (1:250 Millipore, MAB144P) overnight at 4 °C. Samples were washed and incubated with secondary antibodies: goat anti-rat Alexa Fluor 647 (1:500, Thermo Fisher Scientific, A21247), goat anti-mouse Alexa Fluor 647 (1:300, Thermo Fisher Scientific, A28181) or goat anti-rabbit Alexa Fluor 488 (1:500, Thermo Fisher, A27034) for 1 h at RT. Nuclei were labeled with DAPI (1 µg/mL, 5 min, Molecular Probes, Ottawa, ON, Canada) and samples were mounted on microscope slides using Dako mounting medium (Dako, Burlington, ON, Canada). Samples were imaged using a fluorescence microscope. The samples were harvested under a protocol approved by the Montreal Neurological Hospital’s research ethics board (NEU-10-066). Consent was given by all patients and controls (aged 55–76). Tissues were from the cerebral cortex.