Fluorescently tagged anti rabbit or anti mouse secondary antibodies

Cells were grown on glass coverslips in a 24-well plate. Following the indicated treatment, cells were washed twice with PBS and were fixed in a solution of 4% paraformaldehyde in PBS for 20 min. Fixed cells were incubated in blocking solution (0.3% Triton X-100 and 10% normal goat serum in PBS) for 30 min, and then incubated overnight at 4 °C with antibodies against cleaved-caspase 3 (D175, Cell Signaling, Danvers, MA, USA), MAP2 (Hm2, Sigma M9942), Aβ (6E10 SIG39320-200). The cells were then washed three times with PBS and incubated with fluorescently tagged anti-rabbit or anti-mouse secondary antibodies (Invitrogen) in blocking solution for 1 h at room temperature. The cells were then washed twice with PBS and, if indicated, were stained with 0.02% Thioflavin S (Sigma T-1892) for 8 min and then washed three times with 80% ethanol, twice with water and two times with PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma #32670) in PBS for 10 min. All coverslips were then washed with PBS and mounted on microscope slides in an anti-fade medium (Vector Laboratories, Burlingame, CA, USA). Images were acquired using a Zeiss LSM 510 confocal microscope with a ×40 objective. Quantification of the staining intensity and area, and evaluation of co-localization were performed using Fiji software.^{75 (link)}