Te200 fluorescence inverted microscope

Tissue and cells were analysed in a TE200 fluorescence inverted microscope (Nikon), with standard DAPI/FITC/TRITC filters coupled to a colour CCD camera (Evolution, Media Cybernetics) and using the software Image Pro Plus 6.0 for image processing (Media Cybernetics Inc.). Images were alternatively acquired with a TCS-SP5 confocal microscope (Leica).
Quantitative analyses of vascular densities at perilesional areas were performed 1 and 8 WPI. The sections containing the largest cavities for each animal (between 600 and 750 μm deep from the dorsal surface) were identified after immunostaining with anti-GFAP and DAPI of 1 out of each 3 histological sections. The consecutive sections were labelled with RECA-1 and vessels counted within the preserved tissue around the cavities. For quantification of blood vessels bearing BSB, we used SMI-71 to label sections corresponding to 450–600 μm deep from the dorsal surface. We evaluated three animals of each experimental group, in 4–8 distinct fields, photographed at 20 × in the conventional fluorescence microscope (for RECA-1 immunostaining sections) or confocal microscopy (for SMI-71 immunostaining sections). Blood vessel lengths were calculated using Image Pro Plus 6.0 software (Media Cybernetics, Inc.) and AngioTool software as previously described^{71 (link)}.

Tissue and cells were analyzed in a TE200 fluorescence inverted microscope (Nikon), with standard DAPI/FITC/TRITC filters coupled to a color CCD camera (Evolution vf, Media Cybernetics) and using the software Image Pro Plus 6.0 for image processing (Media Cybernetics Inc.). Images were alternatively acquired with a TCS-SP5 confocal microscope (Leica).
Lesion epicenters and the largest diameter of cavity size were determined by digitalizing the cavity contours of GFAP-stained horizontal sections of the dorsal–ventral plane of the injured cord. The analyses were confined to the middle 640 µm, ranging from 120 to 760 µm below the first section from the top of the spine. For quantification, the section with the largest cavity size was chosen as the epicenter and 5 sections in 5 successive slides on each side of this putative epicenter had their areas calculated using the software Image Pro Plus 6.0 (Media Cybernetics Inc.) and plotted against a spatial axis. We evaluated three animals of each experimental group.
GAP-43 and 5-HT fibers were evaluated in conventional fluorescence images and plotted as the sum of the total length of positive fibers, at 3 different regions (injury epicenter; 500 µm rostral and caudal to epicenter), in 3 animals (1 section per animal).

After exposure to normocapnia (5% CO₂) or hypercapnia (20% CO₂) for 24 h, differentiated NHBE cells were fixed with ice-cold 50% acetone/50% methanol for 5 min. Cells were blocked in PBS containing 2% BSA and 0.1% triton X-100 then double-stained with 1:200 polyclonal rabbit anti-human TLR4 antibody (H-80, Santa Cruz Biotechnology) followed by 1:200 Alexa Fluor 555-conjugated goat-anti-rabbit IgG (red) (Invitrogen), and 1:500 monoclonal mouse anti-human acetylated tubulin antibody (Clone 6–11B-1, Sigma) followed by 1:200 Alexa fluor 488-conjugated goat anti-mouse IgG (green) (Invitrogen). Nuclei were identified by staining with 1 µg/ml Hoescht (blue) (Sigma). Images were obtained using a Nikon TE200 inverted fluorescence microscope (Nikon) equipped with a SPOT RT Monochrome Digital Camera (Diagnostic Instruments). All images were captured with the same gain and exposure time using Metamorph software.