2.6. Immunofluorescence

To generate brain sections for immunofluorescence, a subset of db/db and Wt mice from the intrahippocampal TDZD-8 experiments were transcardially perfused with 4% paraformaldehyde, as previously reported (Erion et al., 2014; Wosiski-Kuhn et al., 2014). Brains were cryoprotected and sectioned coronally at 40 micron thickness on a freezing microtome (Leica). Sections were stored at −20 in cryoprotectant before being washed and processed for phospho-tau and total tau immunofluorescence, as reported previously (Stranahan et al., 2011). In brief, sections were quenched in sodium borohydride, blocked in 5% milk, and reacted overnight with mouse monoclonal antibody CP13 (1:200; kind gift of Dr. Peter Davies) and rabbit polyclonal anti-total tau (1:500, Dako Cytomation). The following day, sections were washed and reacted with Alexa fluor-conjugated secondary antibodies (Invitrogen, Molecular Probes, Carlsbad, CA) before being mounted on slides, dried, and counterstained with DAPI. Slides were assigned a numeric code after completion of the immunofluorescence reaction and the code was not broken until completion of the study. Confocal micrographs were acquired through a 25X oil immersion objective on a Zeiss LSM510Meta upright microscope. Micrographs were qualitatively scored, with 0 indicating no phospho-tau labeling, 1 indicating labeling in the CA3 stratum radiatum only, and 2 indicating labeling in the CA3 stratum radiatum and stratum pyramidale.