Neutrophil counting

DM Dylan A. McCreedy
CA Clare L. Abram
YH Yongmei Hu
SM Sun Won Min
MP Madison E. Platt
MK Megan A. Kirchhoff
SR Shelby K. Reid
FJ Frank L. Jalufka
CL Clifford A. Lowell
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Animals were euthanized at 1d post-SCI and 25 µm transverse cryosections were obtained as described above for “White matter sparing”. Every twentieth section (~ 500 µm between sections) was labeled with AlexaFluor647-Ly6G (Clone 1A8, 1:200 dilution, Biolegend), anti-NeuN (1:1000, Millipore), and FluoroMyelin Green (1:300, ThermoFisher Scientific). Sections were then washed and incubated with an AlexaFluor 555-conjugated secondary antibody (1:500, Life Technologies). Images were captured using a Nikon Eclipse upright microscope equipped with a × 10 objective (0.45 NA). Neutrophils were counted using custom Matlab code with regions of interest (ROIs) manually drawn for spinal cord grey matter, white matter, and meninges for each section. The section with the greatest number of neutrophils was designated as the lesion epicenter.

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