Alexa flour 594 conjugated goat secondary antibody

PHrodo-Green-dye–labeled C. albicans (Invitrogen, Carlsbad, CA, USA) were used to access the phagocytosis ability of BV2 cells at the presence or absence of PN. Briefly, 2 × 10⁶ BV2 cells were seeded in each well of 24-well plates. After the cells adhere to the wall, the medium was replaced with serum-free DMEM, and incubated with LPS (100 ng/ml) with or without PN (1 μM) for 24 h. Then the supernatant was replaced by a suspension of pHrodo-labeled C. albicans at a 1:5 ratio (progenitor: yeasts) in 1 ml serum-free DMEM for the indicated durations. At indicated time points, the C. albicans suspension was removed and the cells were rinsed with PBS for 3 times. Then the cells were fixed, permeabilized and blocked, and incubated with primary antibody of F4/80 followed by Alexa Flour 594–conjugated goat secondary antibody (Jackson). The mean green fluorescence intensity of engulfed bacteria was estimated by an epifluorescence microscope (AxioVertA1 and ImagerA2).

Spinal cord tissues or cultured BV2 cells were fixed with precooled 4% paraformaldehyde for 20 min at 4 °C, then permeabilized with 0.2% Triton X-100 for 20 min, and blocked with 10% normal goat serum for 0.5 h. Fixed tissues and cells were then incubated at 4 °C overnight with primary antibodies, followed by Alexa Fluor 488– and Alexa Flour 594–conjugated goat secondary antibody (Jackson ImmunoResearch) for 1 h at room temperature. After triple washing by PBS, nuclei were stained with DAPI (Thermo Fisher Scientific) and fluorescent images were acquired using an epifluorescence microscope (AxioVertA1 and ImagerA2). The lesion volume was obtained by the sum of total lesion according to GFAP⁺ lesion cavity area multiplied by distance (about 200 μm) between the sections.