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The total number of fibers was estimated by multiplying the mean fiber density by the total cross-sectional area of the whole nerve cross section which was measured in the light microscopic analysis.
Approximately 10 high magnification photomicrographs (1000×) were taken from each nerve cross section.
Approximately 300 600 axons were counted in 6 8 micrographs per nerve cross section.
The entire nerve cross sections were photographed at low magnification (200×) and the entire nerve cross section areas were determined.
The counting frame (2500 µm) was systematically placed into the lower right corner of each photomicrograph covering at least one-half of the nerve cross section.
Estimates of the total number of axons were calculated as: total number of axons counted divided by the product of number and area of the counting frames multiplied by the area of the nerve cross section.
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Nerve cross sections did not show any gross alteration either (data not shown).
Myelin damage in optic nerve cross sections was assessed by measuring MBP negative areas, data were displayed as a percentage of the total area.
To quantify the demyelinating phenotype of the different mutants, MBP-negative areas of optic nerve cross sections were determined as measure for demyelination.
Digitized, overlapping pictures (magnification 100×) of semithin sciatic nerve cross sections were fused to a continuous picture of a whole sciatic-nerve cross-section by using Photoshop CS (Adobe systems, San Jose, CA).
Glutaraldehyde-fixed samples of sciatic nerve were processed as described previously (Westaway et al, 1994), with bright-field images of nerve cross sections captured with a 10× objective (Nikon Eclipse 90i microscope).
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