Ix2 ucb dsu spinning disc confocal microscope

For two-dimensional evaluation, 4-μm fresh-frozen sections were stained with immunofluorescent antibodies against desmoglein 2 (DSG2, Epitomics-Abcam, Burlingame, CA) in combination with DAPI (Invitrogen™, Life Technologies, Grand Island, NY). As a negative control, all stainings were performed without primary antibody. Fluorescently labeled species-specific secondary anti–immunoglobulin G (IgG) antibody (Invitrogen™, Life Technologies, Grand Island, NY) was applied for visualization of primary antibody. Images were captured with Olympus IX2-UCB DSU spinning disc confocal microscope (Olympus, Center Valley, PA) with an Evolve electron-multiplying charge-coupled device camera (Photometrics, Tucson, AZ). Dual-color immunofluorescence was performed at 600x magnification and a high–numerical aperture (NA, 1.2) oil-immersion objective. Images were acquired and stored in original SlideBook format (Intelligent Imaging Innovations Inc, Denver, CO). After image capture, files were saved in TIFF format.

For two-dimensional evaluation, 4-μm fresh-frozen sections were stained with immunofluorescent antibodies against CD11B, Olig2, Sox2, CD45 and CMV glycoprotein gB of CMV in combination with DAPI (Invitrogen^™, Life Technologies, Grand Island, NY). As a negative control, all stainings were performed without primary antibody. Fluorescently labeled species-specific secondary anti–immunoglobulin G (IgG) antibody (Invitrogen^™, Life Technologies, Grand Island, NY) was applied for visualization of primary antibody. Images were captured with Olympus IX2-UCB DSU spinning disc confocal microscope (Olympus, Center Valley, PA) with an Evolve electron-multiplying charge-coupled device camera (Photometrics, Tucson, AZ). Dual-color immunofluorescence was performed at 600× magnification and a high–numerical aperture (NA, 1.2) oil-immersion objective. Images were acquired and stored in original SlideBook format (Intelligent Imaging Innovations Inc, Denver, CO). After image capture, files were saved in TIFF format. Images were quantified using image J software. Prior to the measurement of mean FITC background was automatically subtracted from each slide prior assessment of fluorescence. Data was plotted and statistical analyses were conducted on average of mean fluorescence per 5 slides per staining.