Ab6326

Brains were fixed with 4% paraformaldehyde for 24 h, transferred to 30% sucrose for 3 days and stored at – 20 °C in cryoprotectant storage solution until use. Brains were cut into 30-µm coronal sections. Free-floating sections were washed in PBS and incubated in blocking solution (PBS, 5% normal goat serum, 0.2% Triton X-100) for 3 h at room temperature. Sections were incubated with primary antibodies in blocking solution overnight at 4 °C. The primary antibodies used were as follows: ZO-1 (Invitrogen, 61-7300, 1:1000, CA), BrdU (Abcam, ab6326, 1:250, Cambridge, UK), NeuN (Millipore, ABN78, 1:500, MA, USA), 6E10 (anti-Aβ aa 1–16 antibody, Biolegend, SIG39320, 1:500, CA), Iba-1 (Novus Biologicals, NB100-1028, 1:150, CO, USA or Wako, 19-19741, 1:150, VA), CD68 (Bio-Rad, MCA1957, 1:150, CA), and GFAP (Abcam, ab53554, 1:150, MA). After the primary immunoreaction, sections were incubated with Alexa 488 (Invitrogen, A11008, 1:500, CA)  or Alexa 594 (Abcam, A150156, 1:250, MA) conjugated secondary antibodies. For detection of Adu penetration into the brain, brain sections were incubated with anti-human IgG Alexa 555 (Invitrogen, A21433, 1:200, CA). Immunostaining of the sections was visualized with an LSM 700 confocal microscope (Carl Zeiss, Jena, Germany) or an Axio Imager M2 (Carl Zeiss) light microscope. Images were analyzed using ImageJ software (Version 1.52a, NIH, USA).