Detection of AD-relevant biomarkers by IHC

Brain slices were deparaffined and rehydrated before antigen retrieval in citrate buffer (C₆H₅Na₃O₇ •2H₂O 10 mM, pH 6.0) supplemented with Tween-20 0.05%. Brain slices were then incubated with PBS/H₂O₂ 0.7% for inhibition of endogen peroxidases, Streptavidin/Biotin Blocking Kit for endogen biotins and PFBB as described above.

The biomarkers studied and the antibodies used were: cPLA₂-IVA, anti-cPLA₂ [153–166] antibody (5 μg/mL) and the horse anti-goat IgG antibody coupled to biotin at 20 μg/mL in phosphate buffer pH 7.8 containing Tween-20 0.1%; NMDAR, rabbit polyclonal anti-GRIN1 antibody (2.5 μg/mL, MyBiosource) and a goat anti-rabbit IgG antibody coupled to biotin (Vector Labconsult) at 10 μg/mL in phosphate buffer pH 7.8; phosphorylated tau protein, rabbit anti-P-tau (Ser199) antibody (8 μg/mL, Antibodies-Online) and the goat anti-rabbit IgG antibody coupled to biotin (15 μg/mL in phosphate buffer); APP, rabbit anti-AβPP (Amyloid beta A4 Precursor Protein [380–430], 10 μg/mL, Antibodies-Online) and the goat anti-rabbit IgG antibody coupled to biotin (20 μg/mL in 0.1% Tween-20 phosphate buffer). Primary antibodies were prepared in PBS.

The end of the protocol is identical to the previously described one (Vectastain ABC complex, Tris-HCl, DAB). Negative controls by omission of primary or secondary antibodies or by substitution of specific antibodies by non-immune serum in the procedure were used to test the specificity of the IHC. No labeling was detected in the tissue section under such conditions.

The staining obtained on brain slices was quantified similarly as previously described. In this case, the threshold was specifically determined to isolate DAB staining in cell bodies (Supplementary Figure 2).