To examine the degree of neurogenesis (Figure 6H), BrdU (100 mg/kg)

was injected into DG-A::TeTxLC-tau-lacZ and wild-type litters from P15 to P22 (8 days total of injection). Horizontal brain sections from P23 animals were stained with the anti-BrdU antibody as descried above. Mice were euthanized and perfused with 4% PFA/PBS. Their brains were dissected out and postfixed with 4% PFA/PBS for 1 hr at 4°C. One-hundred-micrometer thick horizontal sections were cut on a vibratome and stored in PBS. Sections were placed Abiraterone manufacturer in a drop of PBS on a 10 mm Petri dish and chloromethylbenzamido-DiI (CM-DiI; Invitrogen) coated Elvax (0.1 × 0.1 × 0.1 mm) was placed onto the hilar region of the sections and left for 2 hr at 25°C or for 16 hr at 4°C. After CM-DiI coated Elvax was removed from

the sections, the sections were fixed again with 4% PFA/PBS for 1 hr; blocked in BMS-907351 cell line 2% BSA, 2% goat serum, and 0.1% Triton X-100 in PBS for 1 hr; and incubated with the anti-β-gal antibody at 4°C for 16 hr. Anti-mouse IgG1 Alexa Fluor 488 was used as the secondary antibody. Stained sections were mounted on a slide with Prolong antifade reagent. Images were taken with an Olympus confocal microscope using a 40× lens. For quantification of lacZ staining, color images taken by a digital camera (E990 Nikon) with a dissecting microscope (Olympus) were converted to grayscale images and then inverted with ImageJ software (NIH). The average signal intensity in the stratum lucidum layer of CA3 was calculated with ImageJ.

The average signal intensity in the stratum radiatum of CA3 in the same section was calculated and subtracted as the background. For quantification of β-gal immunostaining with DG lines (Figure 5, Figure 6, and Figure 8), images were acquired on a BX61 microscope (Olympus) with a 20× objective lens using the same exposure time for each experiment. The intensity of staining in the hilar region of the hippocampus was analyzed with ImageJ software. The intensity in the neighboring area without stained axons (in the stratum radiatum of CA3) in the same section was calculated and subtracted as the background Oxygenase (see Figure S3B). Mice were anesthetized with ketamine/xylazine (60 mg/kg ketamine, 10 mg/kg xylazine) and placed in a hand-made frame, and their skulls were exposed. A hole was generated with a 26G needle (−1.2 mm from bregma, 1.0 mm lateral from the midline, and 1.5 mm ventral from the skull surface for EC::TeTxLC-tau-lacZ mice; −2 mm from bregma, 1.5 mm lateral from the midline, and 2 mm ventral from the skull surface for DG::TeTxLC-tau-lacZ mice) onto the left hemisphere. Optimal locations were determined by preliminary experiments with Trypan Blue injections. TTX (0.5 μl of 5 μM TTX in PBS) was injected every 24 hr from P9 to P12, P14, or P16 (4, 6, or 8 days total of injection) with a Hamilton syringe (75RN, 7762-03) mounted on a micromanipulator. To suppress neurogenesis, AraC was injected either i.c.v. or i.p.