Mice were perfused with fresh 1× PBS and brains were extracted and subsequently fixed in a glass vial of 4% paraformaldehyde for 48 h. After fixing, a Leica VT1000 S vibratome was used to partition the tissue into 50 μm coronal sections and stored chronologically in a specimen plate with PBS containing 0.02% sodium azide. Coronal brain sections including the hippocampus underwent immunohistochemistry as previously described [46 (link), 48 (link)] with the appropriate antibodies. Rbbp7 and AT8 images were taken at a resolution of x = 512, y = 512, and z = 1 using a 60× oil immersion objective and tau acetylation, p300 and GFP images were taken at a resolution of x = 1024, y = 1024, and z = 1 using a 40× oil immersion objective. A zoom of 1.5 was used and images were taken with the Leica DM2500 confocal microscope. Quantification was performed by taking signal area and intensity using Image J software. To quantify AT8- and AT100-positive cell counts, images from all animals were taken with a Zeiss Axio Imager A1 using a 5× objective. Images were photomerged to rebuild the image, and AT8- and AT100-positive cells numbers were obtained using imageJ. The experimenter was blinded to the group allocation.
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