Ab175262

For western-blots we used rabbit polyclonal antibodies against the synthetic peptide within 29–59 N-terminal aa’ of human TREM2 (1:200, Abcam, ab175262, Cambridge, MA) (Ma et al., 2016 (link); Raha et al., 2016 ) and against to the C-terminus of Iba1 (1:500, Wako, Osaka, Japan). TREM2 antibody recognized non-glycosylated/immature (~ 25 kDa) and glycosylated/mature (~ 50 kDa) form (Fig. S1). Loading control was a β-tubulin antibody (1:1000, Millipore, Billerica, MA) (Mufson et al., 2012 (link); Perez et al., 2015 (link)). Aβ plaques and microglia quantitation employed a mouse monoclonal IgG antibody against human APP/Aβ (6E10) (1:2000, BioLegend, San Diego, CA) and a rabbit polycloncal antibody against Iba1 (1:1000, Wako), respectively.

Briefly, proteins were denatured in SDS loading buffer to a final concentration of 5 mg/ml. Proteins (50 μg/sample) were separated by 4–20% SDS-PAGE (Lonza, Rockland, ME) and electrophoretically transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore) (Perez et al., 2015 (link))]. Membranes were blocked in Tris-buffered saline (TBS)/ 0.05% Tween-20/ 5% milk (1 h) at room temperature (RT). TREM2 or Iba1 antibodies were added to blocking buffer and membranes were incubated overnight (4^o C), washed, incubated at RT (1 h) with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:8000, Bio-Rad, Hercules, CA) or goat-anti rabbit IgG secondary antibody (1:5000, Bio-Rad, Hercules, CA), visualized by chemiluminescence on a Kodak Image Station 440CF (Perkin-Elmer, Wellesley, MA) and quantified with Kodak 1. The ~ 25 kDa TREM2 band was quantified across clinical groups. Protein signals were normalized to β-tubulin and analyzed in three independent experiments (Perez et al., 2015 (link)). Immunoblot controls consisted of pre-adsorption of the TREM2 antibody with an excess of the immunogenic TREM2 peptide (Abcam, ab217864), that blocks the anti TREM2 antibody-N-terminal used in the study (Abcam, ab175262) and deletion of the primary, both resulting in the absence of immunoreactive bands in the membranes (see Fig. S1).