Eclipse ti c2 inverted confocal microscope

Myelin-engulfing macrophages were assessed in sections with the most intense IgG deposition (−1200, −600, and +600 μm from the injury epicenter). To this end, the selected sections were co-stained for fluoromyelin and CD11b. First, sections were stained for CD11b (primary + secondary antibody) as described above. Next, following washes, sections were incubated with fluoromyelin (1:100, Life Technologies, F34652) and DAPI (1:250) in 5% goat serum + 1% BSA + 0.3% Triton X-100 in PBS, overnight. Images were acquired with a Nikon eclipse Ti C2+ inverted confocal microscope under identical settings for each marker. The area of positive immunofluorescence for each marker was estimated with ImageJ and expressed as % of the total DAPI+ area of the spinal cord section. Here, we report averaged values that were obtained from three sections (representing distances −1200, −600, and +600 μm from the injury epicenter) per animal.

Complement fixation by deposited IgGs in the spinal cord was assessed in spinal cord sections with the highest IgG signal. These sections, which were located −1200, −600, and +600 μm from the injury epicenter, were co-stained for IgG and complement C3b protein and the amount of complement fixing IgGs was semi-quantified based on the IgG+/C3b+ positive immunofluorescent area. Briefly, sections were first stained for IgG as described above. Next, they were incubated with anti-mouse C3b-FITC antibody (1:100, Cederlane, clone 11H9 recognizing C3/C3b/iC3b) and DAPI (1:250) diluted in 5% goat serum + 1% BSA + 0.3% Triton X-100 in PBS, overnight at 4°C. Lastly, sections were washed, cover-slipped and imaged with a Nikon eclipse Ti C2+ inverted confocal microscope, as described above. For each section, the area of positive immunofluorescence from IgG alone, C3b alone and IgG and C3b signal was estimated with ImageJ and expressed as % of the total area of the spinal cord section. Then, the obtained values for the three sections (representing distances −1200, −600, and +600 μm from the injury epicenter) were averaged per animal.