Eclipse te300c2 confocal laser scanning

After two days of transfection with the chimeric constructs HA-APP-mGFP and HA-Bace1-mBFP, cells were washed with phosphate buffered saline (PBS, 0.5 mM MgCl₂, 0.8 mM CaCl₂, pH 7.4) and fixed in 4% (w/v) buffered paraformaldehyde (PFA) for 12 minat room temperature. Then, cells were blocked with a 30 min incubation with 4% bovine serum albumin (BSA) and incubated for 30 min with 1:500 diluted primary rabbit anti-HA antibody (ab9110 Abcam, UK). After incubation, cells were carefully washed and incubated for 30 min with 1:500 diluted secondary goat anti-rabbit Alexa Fluor 568 antibody (Abcam, UK). Coverslips were washed with PBS and water and mounted on a glass slide. Cell imaging was performed on a Nikon Eclipse TE300C2 confocal laser scanning (CLSM) (Nikon, Japan) equipped with a Nikon 60x immersion oil objective (Apo Plan, NA 1.4) and with Coherent CUBE (diode 405 nm), Melles Griot (Argon 488 nm) and Coherent Sapphire (Sapphire 561 nm) lasers. Emission filters for imaging were 452/45 nm (for mBFP), 514/30 nm (for mGFP) and 595/60 nm (for Alexa 568). Settings were maintained constant for each analysis. > 10 cells were analyzed for each examined sample by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).