Dephosphorylation of AD Tissue

Ethanol-fixed, paraffin-embedded AD tissue was sectioned and rehydrated followed by citric acid antigen retrieval as described above. Tissue was enzymatically dephosphorylated by calf intestinal phosphatase (CIP) (M0290S, New England BioLabs, Ipswich, MA) diluted to 33 U/mL in 50 mM Tris pH 7.6, 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT and incubated for 2 hours at 37°C. Tissue was rinsed in 50 mM Tris pH 7.2 and sequentially dephosphorylated with λ-phosphatase (New England BioLabs P0753S) at 650 U/mL in PMP buffer (New England BioLabs B0761S) with 1 mM MnCl₂.