Computational protocol: Dynamic Changes in Local Protein Synthetic Machinery in Regenerating Central Nervous System Axons after Spinal Cord Injury

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Protocol publication

[…] Imaging was performed using a confocal microscope (Zeiss LSM 700) and Zen software (Zeiss). All image acquisition parameters such as laser power, pinhole, PMT gain/offset, and pixel dwell time were matched within individual markers of PSM between the experimental groups. A PNG section from each rat reacted with all reagents except a primary Ab which was used as a reference for acquisition of background signals. Imaging parameters were set below those producing no signal with primary Ab control. Images were obtained at 63x magnification as three-dimensional z-stacks of 100 × 300 μm in area generated by automated stitching of three individual 100 × 100 μm tiles. z-stacks were acquired at 0.3 μm step interval between planes with a total of 10–15 planes per stack spanning a depth of 3.0–4.5 μm. The tile scans were taken at two comparable locations within the distal third of each nerve section and a total of 3 sections were analyzed per rat.To quantify the intensity of intra-axonal signal, each z-stack was resolved into individual XY planes containing both neurofilament staining as an axonal marker and one of the markers of PSM. The RG2B plug-in (https://imagej.nih.gov/ij/plugins/rg2bcolocalization.html) on NIH ImageJ (https://imagej.nih.gov/ij/) was used to extract the pixels that colocalized between neurofilament stain and PSM markers. This was performed for each plane of the stack to isolate pixels representing axonal signal. The pixel intensity from each plane was then normalized to the neurofilament area in corresponding planes to account for the variability of number of axons per tile scan. The resultant values for each plane were averaged to get a mean value per z-stack. […]

Pipeline specifications

Software tools Stitching, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Spinal Cord Injuries