Pp2b ppp3ca

Immunohistochemistry was performed as previously described.^{23 (link)} Paraffin-embedded tissues were sectioned in a coronal plane at 10–20 μm. The tissue sections were rehydrated, blocked with blocking solution (1% H₂O₂), and incubated with p-Tau (S202/T205) (1:200), PP2B/PPP3CA (1:200; SantaCruz Biotech), p-Tau (S199) (1:200; Abcam) and anti-III tubulin antibody (1:500 dilutions; Sigma) for 24 h. After washing three times, the slides were processed with Vector ABC Kit (Vector Lab). The immunoreactive signals were developed with DAB chromogen (Thermo Fisher Scientific, Meridian, Rockford, IL, USA) and analyzed under a bright field microscope.

Tissues and cells were immunostained for p-Tau (S199) (rabbit polyclonal, 1:200; Abcam, Cambridge, UK), p-Tau (S202/T205) (mouse monoclonal, 1:500; Thermo Scientific, Waltham, MA, USA) and PP2B/PPP3CA (rabbit polyclonal, 1:200; SantaCruz Biotech, Dallas, TX, USA) according to a previous report.^{22 (link)} For confocal microscopy, the specimens were incubated for 1 h with fluorescence (FITC)-conjugated secondary antibody (Vector, Burlingame, CA, USA) and Cy3-conjugated secondary antibody (Jackson Lab, West Grove, PA, USA) after the primary antibody incubation. The images were analyzed using a Spinning Disk Confocal microscope (IX2-DSU, Olympus, Tokyo, Japan). Pre-absorption with excess target protein or omission of primary antibody was used to demonstrate antibody specificity and determine the background generated from the detection assay.