Obt0030

Mice were deeply anesthetized and transcardially perfused with ice‐cold 0.9% saline, followed by 4% paraformaldehyde dissolved in 0.1 M phosphate‐buffered saline (PBS). The fixed, cryoprotected mouse brains were frozen and then cut into 35‐μm‐thick coronal sections. Free‐floating sections were washed three times in PBS, following which they were incubated for 1 hr in 5% normal goat serum in PBS containing 0.3% Triton X‐100. Sections were incubated overnight at 4°C with the following primary antibodies: 6E10 mouse monoclonal anti‐Aβ (1:1,000, Chemicon; MAB1560), rat monoclonal anti‐bromodeoxyuridine (BrdU; 1:100, AbD Serotec; OBT0030), goat anti‐doublecortin (DCX; 1:200, Santa Cruz Biotechnology; SC8066), mouse anti‐glial fibrillary acidic protein (GFAP; 1:200, Abcam; AB4648), and mouse monoclonal anti‐BDNF (1:500, Abcam; ab203573) antibody. After washing three times in PBS, the sections were incubated with the corresponding Invitrogen Alexa Fluor secondary antibodies in PBS for 1 hr at room temperature. The sections were then washed and mounted, and coverslips were secured on the slides using a mounting medium (Vector Laboratories, Inc. Burlingame, CA; H1200). Confocal images were captured using a Nikon confocal Axiovert microscope (Carl Zeiss, Göttingen, Germany; LSM510).

BrdU immunofluorescent labeling was obtained with a 1:500 dilution of the rat anti-BrdU antibody (Accurate Chemical and Scientific Corporation OBT0030, Westbury, NY, USA) and doublecortin (DCX) immunohistochemical labeling was obtained with a 1:800 dilution of the goat anti-DCX antibody (SantaCruz Biotechnology, Dallas, TX, USA). Immunolabeling for both antibodies followed previous descriptions (Naninck et al., 2015 (link); Hoeijmakers et al., 2018 (link)).

Free-floating sections were rinsed in PBS for 30 min. Then, the sections were incubated in 2N HCl for 60 min at 37°C. Afterward, the sections were rinsed in PBS for 30 min and then blocked in PBS containing 0.5% Triton-X-100 and 3% donkey serum for 30 min. The sections placed in monoclonal rat anti-BrdU (1:1000, OBT0030; Accurate, RRID: AB_2313756), monoclonal goat anti-DCX (1:200, sc-8066, Santa Cruz Biotechnology, RRID: AB_2088494), and monoclonal mouse anti-NeuN (1:250, MAB377, Chemicon, RRID: AB_2298772) at 4°C for 48 h. Sections were then rinsed in PBS for 30 min and incubated in Alexa Fluor 488 donkey anti-rat (1:250, A-21208, Invitrogen, RRID: 2535794), Alexa Fluor 555 donkey anti-goat (1:250, A-21432, Invitrogen, RRID: AB_2535853), and Alexa Fluor 647 donkey anti-mouse (1:250, A-31571, Invitrogen, RRID: AB_162542) for 2 h at room temperature. Following a 30-min rinse in PBS, all sections were mounted onto gelatin-subbed slides, dehydrated, and coverslipped under PVA-DABCO mounting media (Figures 1G–J).