Alexa fluor 555 anti rabbit antibody

Fly brains were fixed in 4% paraformaldehyde (PFA) prepared in PBST (PBS + 0.5% Triton X‐100) for 3 h at room temperature. The samples were washed thrice with PBST for 10 min and incubated in blocking solution (2% normal goat serum + 2% bovine serum albumin (BSA) + 0.5% Triton X-100) for 3 h. Then, the samples were incubated with anti-Aβ42 antibody prepared in the blocking solution for 48 h at 4°C (blocking solution 1 : 200; Santa Cruz, CA, USA). Alexa Fluor 555 anti-rabbit antibody (PBST 1 : 200; Cell Signaling Technology, Danvers, USA) was used as the secondary antibody.

HEI-OC1 cells were cultured in 24-well dishes with DMEM plus 10% FBS. Briefly, the cells were fixed with 4% paraformaldehyde for an hour and then permeabilized with 0.25% Triton X-100 for 30 min. After that, the cells were blocked with 1% BSA in PBS for an hour and then incubated overnight with an LC3-B primary antibody (#2775, 1:1000; Cell Signaling Technology, Danvers, MA, USA) and a 4-hydroxynonenal primary antibody (#BS-6313R, 1:200; Bioss, Woburn, MA, USA) at 4 °C. After three washes with PBS, the cells were stained with an Alexa Fluor-555 anti-rabbit antibody (#4413, 1:2000; Cell Signaling Technology, Danvers, MA, USA), Hoechst (#33258, Sigma, St. Louis, MO, USA), and LipiDye (#FDV-0010, Tokyo, Japan) for 10 min in the dark. The specimens were then observed under a laser scanning confocal microscope (ZEISS LSM-800, Jena, Germany).