Double-labeling immunofluorescence

To examine the co-localization of Aβ43 and Aβ42, double-labeling immunofluorescence was performed. The sections were deparaffinized and rehydrated, and antigen retrieval was performed using formic acid. After being incubated with the primary antibodies at 4°C overnight, the sections were washed with TBS. As secondary antibodies, Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100, Life Technologies, Eugene, OR, USA) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:100, Life Technologies) antibodies were mixed in TBS. The sections were incubated with secondary antibodies at 37°C for 1 hr and then mounted with Vectashield (H-1500, Vector Laboratories, Burlingame, CA, USA). The immunolabeled antigens were visualized using a Carl Zeiss LSM700 Confocal Laser Scanning Microscope (Carl Zeiss, Tokyo, Japan).