Trem2

Single cell suspensions were incubated with an anti-FcγIII/II receptor antibody (clone 93) to block unspecific binding and Zombie Violet^TM (Biolegend, Germany), a fixable viability dye. Thereafter, cells were stained with fluorochrome conjugated antibodies against cell surface markers: CD45 (30-F11, eBioscience, Germany), CD11b (M1/70, eBioscience, Germany), Ly6G (1A8, BD Biosciences, Germany), Ly6C (HK1.4, eBioscience, Germany), CCR2 (475301, R&D, USA), TREM2 (237920, R&D, USA), CD36 (HM36, Biolegend, Germany), in FACS buffer (PBS containing 2 % FCS and 0.1 % NaN₃) for 30 min on ice and then washed and fixed in 4 % paraformaldehyde (PFA) for 10 min.

Equal amounts of protein (30 μg) were separated by electrophoresis in NuPAGE Bis-Tris Gels (Life Technologies) and transferred to nitrocellulose membranes. The membranes were hybridized with various primary antibodies (DYRK1A 7D10 (1/250, Abnova), DYRK1A D1694 (1/500, Sigma), GFAP (DAKO, 1/4000), Vimentin (1/1000, Abcam), pSTAT3(Y705) (1/1000, Cell Signaling), STAT3 (1/1000, Cell Signaling), IBA1 (1/2000, Wako), CD68 (1/1000, BioLegend), IDE (1/200, Santa Cruz), TREM2 (R&D Systems, 1/500), GAPDH (1/1000, Abcam)). Various secondary antibodies were also used (ECL Anti-mouse horseradish peroxidase linked, 1/2000, GE Healthcare; ECL Anti-rabbit horseradish peroxidase linked, 1/2000, GE Healthcare). Membranes were developed using enhanced chemiluminescence (Thermo Fisher Scientific). Signals were detected with Fusion FX7 (Vilber Lourmat) and analyzed and quantified using ImageJ (NIH).

Brain sections (40 μm) were cut using a vibratome and stained using modifications of standard procedures previously described [33 (link)]. Staining was performed in a limited number of batches that were balanced across experimental groups. For immunohistochemistry, sections were subjected to heat-mediated antigen retrieval with 10 mM EDTA for 10 min at 95 °C. Endogenous peroxidases were blocked by 3% H₂O₂ and 10% methanol in Tris-buffered saline (TBS, 30 min at room temperature). Sections were permeabilized in 0.1% Triton X-100 for 15 min, blocked by a 30 min incubation in blocking buffer (TBS with 3% bovine serum albumin and 0.1% Triton X-100), followed by incubation at 4 °C with primary antibodies (diluted in blocking buffer) against the microglial marker ionized calcium binding adaptor molecule 1 (Iba1) (WAKO rabbit; 1:500 dilution) and/or TREM2 (R&D Systems; sheep 1:500 dilution) for 2–3 days. After washing, sections were incubated with Alexa fluorophore-conjugated secondary antibodies (Invitrogen; anti-rabbit and anti-sheep) diluted 1:500 in blocking buffer for 1–2 days. To label amyloidogenic plaques, immunostained sections were incubated with 0.5% Thioflavin S (ThioS; Sigma-Aldrich) for 10 min and subsequently washed sequentially with 70% ethanol, 50% ethanol, and purified H₂0 before mounting on glass slides with VECTASHIELD® Antifade mounting media (Vector Labs).