Goat anti trem2

Western blotting was performed as previously described [6 (link)]. Briefly, after brain protein sample preparation using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA), equal amounts of protein were loaded on an SDS-PAGE gel and run using electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked and incubated overnight at 4 °C with the following primary antibodies: goat anti-TREM2 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-PI3K (1:1000, Cell signaling, Danvers, MA, USA), rabbit anti-phosphorylated Akt (p-Akt, 1:1000, Cell signaling), rabbit anti-Akt (1:1000, Cell signaling), rabbit anti-TNF-α (1:1000, Abcam), rabbit anti-IL-1β (1:1000, Abcam), anti-Bcl-2 (1:2000, Abcam), anti-Bax (1:4000, Abcam), and goat anti-β-actin (1:5000, Santa Cruz Biotechnology). Appropriate secondary antibodies (1:3000, Santa Cruz; 1:5000, Abcam) were selected to incubate with the membrane for 2 h at room temperature. The bands were probed with an ECL Plus chemiluminescence regent Kit (Amersham Biosciences, Arlington Heights, PA, USA) and visualized with the image system (Versa Doc, model 4000, Bio-Rad, Hercules, CA, USA). Relative density of the protein immunoblot images were analyzed by ImageJ software (ImageJ 1.4, NIH, Bethesda, MD, USA).

After antigen retrieval step (45 minutes) and block of non-specific binding (1 hour), paraffin-embedded tissue sections were incubated at 4°C (overnight or for 1hour) with rabbit polyclonal anti-SLC16A1 Ab (1:300, Bethyl), mouse monoclonal anti-SLC16A3 (1:100, Santa Cruz), polyclonal sheep anti-CD90 (1:100, R&D), biotin anti-CD68 (1:100, Novus), goat anti-TREM2 (1:100, Abcam), mouse anti-PRG4 (1:100, Novus), and rabbit anti-CLIC5 (1:100, ThermoFisher). Streptavidin Alexa Fluor™ 594, donkey anti-Sheep IgG Alexa Fluor™ 546, donkey anti-Rabbit IgG Alexa Fluor™ 488, donkey anti-goat IgG Alexa Fluor™ 546, donkey anti-Mouse IgG Alexa Fluor™ 647 were used as secondary antibodies (1:300). Hoechst was used for nuclear staining. Slides were mounted with Prolong Gold Antifade reagent (Invitrogen). Images were acquired on confocal microscope (Zeiss LSM 780). Colocalization was measured by calculating the Pearson’s correlation coefficient using ImageJ software as described (21 (link)).

Briefly, after mice were anesthetized with isoflurane and intracardially perfused with ice-cold PBS and 10% formalin, the brains were removed, fixed in 10% formalin overnight at 4 °C, and dehydrated with 30% sucrose for 3 days. Brain tissues were snap-frozen at − 80 °C and cut into 10-μm-thick coronal sections using a cryostat (CM3050S; Leica Microsystems, Bannockburn, III, Germany). Immunofluorescence staining was performed as previously described [7 (link)]. Brain samples were incubated overnight at 4 °C with primary antibodies including rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:100, Abcam), rabbit anti-glial fibrillary acidic protein (GFAP, 1:100, Abcam), rabbit anti-neuronal specific nuclear protein (NeuN, 1:200, Abcam), rabbit anti-myeloperoxidase (MPO, 1:500, Abcam), and goat anti-TREM2 (1:1000, Abcam). The sections were then incubated with corresponding secondary antibodies (1:200, Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature, followed by visualization using a fluorescence microscope (Leica Microsystems).