Anti human trem2

Free-floating brain sections (40 μm thickness; coronal) were collected from the same brain region for all the animals, and brain regions were confirmed under the microscope by comparing sections to images on the Allen Mouse Brain Atlas (http://mouse.brain-map.org). The tissue was blocked in 5% goat serum for 1 h and incubated with the primary antibodies overnight. Sections were then incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at room temperature (1:1,000; Invitrogen). The following primary antibodies were used: anti-human TREM2 (16E1, produced in-house at Mayo Clinic, 1:500); anti-IBA1 (Cat# 019-19741, 1:1,000; Wako); anti-GFAP (Cat# Pu020-UP, 1:1,000; BioGenex); anti-NeuN (Cat# MAB377, clone A60, 1:1,000; Millipore); anti-Aβ (MOAB-2, Cat# ab126649, 1:1,000; Abcam); anti-LAMP1 (Cat# ab25245, 1:500; Abcam); anti-CD68 (Cat# MCA1957, 1:300; VWR); and anti-CD11c (Cat# 14011482, 1:200; Thermo Fisher Scientific). To identify fibrillar Aβ plaques, free-floating sections from 5xFAD/TREM2 mice were permeabilized with 0.25% Triton X-100 in PBS and stained with 10 µM X-34 (Cat# SML1953; Sigma-Aldrich) in 40% ethanol + 0.02 M NaOH in PBS as described (Ulrich et al., 2018 (link)). Researchers were blinded to genotypes and groups when performing and quantifying the immunofluorescence staining.

p38-MAPK inhibitor (SB203580), ERK1/2 inhibitor (U0126), JNK inhibitor (SP600125), NF-κB inhibitor (Bay 11–7082), Akt inhibitor (LY-294002), and DAPI were purchased from Sigma-Aldrich. GM-CSF was purchased from R&D Systems. Kits for Cell Proliferation Assay (MTS), DeadEnd Fluorometric TUNEL System, and TEV protease were purchased from Promega. In Situ Cell Death Detection kit and cOmplete His-Tag Purification Resin were purchased from Roche. BrdU ELISA kit was purchased from Abcam. Protein A Agarose and Alexa Fluor 488 Phalloidin were purchased from Thermo Fisher Scientific. Antibodies for Western blotting used in this study, including anti–Phospho-NF-κB p65 (Ser536), anti–total-NF-κB p65, anti–Phospho-Akt (Ser473), anti–total-Akt, anti-Phospho-GSK3β (Ser9), anti–total-GSK3β, anti–β-catenin, and anti–β-actin, were purchased from Cell Signaling Technology; anti–human TREM2 antibody for Western blotting was purchased from R&D Systems (AF1828). Antibodies for immunofluorescence including anti-Iba1 and anti–human TREM2 were purchased from Wako and Santa Cruz Biotechnology (sc-373828), respectively.