Latex beads rabbit igg fitc complex

Microglial phagocytosis capacity was measured using an in vitro assay adapted from (Fonken et al., 2018 (link)). Briefly, isolated microglia (described above) from amygdala and hippocampus of young and aged rats fed chow or HFD were plated at a density of 1.2 × 10⁴ cells (amygdala) or 2 × 10⁴ cells (hippocampus) per well in eight-well chambered culture slides (Nunc Lab-Tek II; ThermoFisher Waltham, MA, USA). The microglia were incubated at 37 °C, 5% CO₃ for 2 hr. Latex beads-rabbit IgG-FITC complex (1:100 final concentration on cells; Cayman Chemicals, Ann Arbor, MI, USA) were then added to the wells. After 30 min incubation, the beads were washed off with PBS and the cells were fixed with cold 4% paraformaldehyde for 30 min. The slides were stored in PBS. For immunocytochemistry, slides were incubated in 10% normal donkey serum for 30 min, then with primary antibody (rabbit anti-Iba1, 1:1000; Wako) overnight. Cells were washed three times with PBS, then incubated with secondary antibody (Alexa 546 donkey anti-rabbit, 1:500; ThermoFisher) and DAPI for 2 hr. Slides were coverslipped and imaged using an inverted Nikon Ti-E microscope (at the CU-Boulder Light Microscopy Core Facility). For image capture and analysis, 20 images per well were taken (random areas within the well) and we assessed the percentage of microglial cells containing green beads.