Sixteen-micrometer horizontal sections were cut on a cryostat and stored at −80°C until use. Sections were blocked in 2% BSA, 2% goat serum, and 0.1% Triton X-100 in PBS for 1 hr, followed by the incubation with primary antibodies at 4°C for 16 hr in the same solution. Sections were washed in PBS and secondary antibodies were applied in PBS for 1 hr. Nuclei were stained with

DAPI. Antibodies and dilutions used were: β-gal (Sigma, 1:500), NeuN (Chemicon, 1:1000), calretinin (Millipore, 1:500), DCX (Santa Cruz, 1:200), Ki67 (Thermo scientific, 1:200), VGLUT1 (Millipore, 1:4500), and bassoon (Assay Designs, 1:500). Stained sections were observed with an epifluorescence or confocal microscope (Olympus). Hippocampi were isolated from P11–12 (EC lines) or P15–17 (DG selleckchem lines) mice, and 400 μm transverse slices were cut using a tissue chopper. Slices were then allowed to recover in an interface chamber for a minimum of 2 hr at 25°C. For field recordings, slices were placed in a chamber and perfused constantly with oxygenated artificial cerebral spinal fluid (aCSF) heated to 27°C–28°C. aCSF contained (in mM)

119 NaCl, 2.5 KCl, 1 Na2PO4, 26.3 NaHCO3, 11 glucose, 1.3 MgSO4, and 2.5 CaCl2. To obtain input-output curves, fEPSPs were stimulated using a cluster electrode (FHC). For the EC line recording, both the stimulating electrode and the recording electrode (containing 3 M NaCl) were placed in the molecular layer of the DG. For the DG line experiments, the stimulating electrode was placed in the hilus of the DG and the recording electrode was placed in the stratum radiatum of CA3. Responses were collected with www.selleckchem.com/products/E7080.html also a MultiClamp 700B amplifier (Axon Instruments) and analyzed using

Clampfit software (Axon Instruments). Statistical significance was determined using a two-way ANOVA. To examine DG cell survival (Figures 4E and 4F), wild-type and DG-A::TeTxLC-tau-lacZ mice (15 mice each) received a single injection of BrdU (300 mg/kg) at P7–8. Mice were perfused with 4% PFA/PBS at P15, P20, and P25 (five mice for each day per each genotype). Their brains were postfixed with 4% PFA/PBS for 16 hr, cryoprotected in 30% sucrose, and frozen in the OCT embedding compound. Fifty-micrometer horizontal sections were cut with a cryostat and placed in PBS (floating). Every sixth section was incubated with 2 M HCl for 30 min at 37°C, washed in 0.1 M Tris buffer (pH 8.0) for 10 min, and washed three times in PBS for 3 min. Sections were then blocked in 2% BSA, 2% goat serum, and 0.1% Triton X-100 in PBS for 1 hr, and incubated with the anti-BrdU (rat monoclonal; 1:400 Chemicon) and NeuN antibody at 4°C for 16 hr. After washing in PBS, sections were incubated with the goat anti-rat Alexa Fluor 488 and goat anti-mouse IgG1 Alexa Fluor 568 secondary antibodies for 1 hr. Sections were washed again in PBS and mounted on slides with Prolong antifade reagent (Invitrogen).