Lsm 880 airyscan super resolution confocal microscope

CD68 expression levels were assessed using immunofluorescence and confocal microscopy. Briefly, irradiated and control HMC3 cells were cultured in 4-well CELLview^™ dishes (Greiner Bio-One North America). Cells were fixed with 4% paraformaldehyde (PFA) for 15 min, and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min. Cells were incubated with mouse anti-human CD68 (1:100) overnight at 4°C. Then, cells were incubated with Alexa Fluor-647-labeled goat anti-mouse IgG (1:500) at RT for 2 h. Images were captured using an LSM 880 Airyscan super-resolution confocal microscope (Carl Zeiss, Oberkochen, Germany). Mean fluorescence intensity (MFI) was quantified using ImageJ (NIH).

HMC3 cells were plated in 4-well CELLview^™ dishes (Greiner Bio-One North America) and cultured at 37°C with 5% CO₂. Immunofluorescence was performed at 1 h after IR to determine NF-κB p65 nuclear translocation. Briefly, cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100. After blocking with 5% normal serum, cells were incubated with p65 antibody (1:100) over night at 4°C. After washing with PBS, cells were incubated with goat anti-rabbit IgG Alexa Fluor 647 (1:500) at RT for 2 h. Images were acquired using an LSM 880 Airyscan super-resolution confocal microscope (Carl Zeiss, Oberkochen, Germany). Mean fluorescence intensity (MFI) was measured using ImageJ (NIH).

CD68 expression levels were assessed using immunofluorescence and confocal microscopy. Briefly, HMC3 cells were cultured in 4-well CELLview^™ dishes (Greiner Bio-One North America) and treated with Aβ₄₀ or Scr-Aβ₄₀ for 48 h. Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. Next, cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min and then incubated with blocking buffer (5% goat serum and 0.1% Triton X-100 in PBS) at RT for 1 h. Cells were incubated with mouse anti-human CD68 (1:100, ThermoFisher Cat# 14-0688-82, clone: KP1) and rabbit anti-Iba1 (1:500, FUJIFILM, #019–19741) antibodies overnight at 4°C. Then, cells were incubated with Alexa Fluor-488-labeled goat anti-rabbit (1:500, Invitrogen, #A11008) and Alexa Fluor-647-labeled goat anti-mouse (1:500, Invitrogen, #A32728) secondary antibodies at RT for 2 h. Nuclei were counterstained with 1 μg/mL DAPI, and cells were mounted with ProLong Gold anti-fade reagent (Invitrogen, Cat# 36930). Images were captured and analyzed using an LSM 880 Airyscan super-resolution confocal microscope (Carl Zeiss, Oberkochen, Germany). Mean fluorescence intensity (MFI) was quantified using ImageJ (NIH).