Axiophot fluorescent microscope

BV2 or primary microglial cells were seeded at a density of 25 000 cells per coated-glass coverslips. Fluorescent red-latex beads (mean particle diameter 1 µm, Sigma) were preopsonized in 50% FBS in sterile PBS for 1 h and loaded to the cells at concentrations of 50 beads per cell for 4 h at 37ºC. After that, remaining beads were washed off the cells twice and cells were fixed by 4% paraformaldehyde in PBS for 15 min at room temperature and mounted in glass slides with FluorSave mounting medium. Alternatively, cells were incubated for 4 h with 100 nM oligomeric 1-42 amyloid β-peptide. For preparation, dry Aβ peptide (Anaspec) was dissolved in alkaline conditions and further incubated at 37ºC for 12 h for oligomerization (Berrocal, Sepulveda, Vazquez-Hernandez, & Mata, 2012) (link). Then, phagocytized peptide was identified by immunostaining with anti-Aβ antibody (1:500). Immunostaining with anti β-tubulin
(1:500), the rat monoclonal CD68 (1:500, AbD Serotec), and nuclear staining with DAPI were also used for further analysis. Images were captured with the Zeiss Axiophot fluorescent microscope or with a Leica TCS SP5 confocal microscope to obtain orthogonal projections and verify that phagocytosed particles were inside the cells.