2.6.5. Immunohistochemistry of Phosphorylated Tau

The immunohistochemistry protocol for pTau was described elsewhere [23,24,25,26,27,28]. Briefly, the paraffin sections were first deparaffinized and rehydrated with graded alcohol. Then the sections were washed with PBS-Triton-X-100 (0.5%, PBS-T) for 5 min and blocked with 10% goat serum (dissolved in PBS-T) for 30 min at room temperature with gentle shaking. The sections were incubated with pTau antibody (mouse monoclonal, 1:200), overnight on the shaker. On the next day, the sections were washed three times, 5 min each, and incubated with anti-mouse secondary antibody, conjugated with Alexa-488, for 1 h at room temperature with gentle shaking. Then the sections were washed with water, three times, 5 min each, followed by counterstaining with PI (500 nM) for 5 min. Finally, the sections were washed, and mounted with anti-fading mounting media and the images were taken using fluorescent microscopes (Leica, Wetzlar, Germany) with appropriate excitation and emission filters. The bright green signal was considered as pTau-positive cells. The number of pTau-positive cells were counted manually from the spleen tissue of three mice in each group with at least 15–20 randomly captured images and expressed as mean pTau positive cells/microscopic field.