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The number of immune-reactive cells against the neuronal marker was counted and calculated on the basis of DAPI (Molecular Probes, Eugene, OR) stained total immunoreactive cells within the area.
The appearance of each marker was counted for each dataset and algorithm.
The total cell number and total number of positive cells for each marker was counted in each histologic zone by two different readers, and the percentage of positive cells was calculated in each field.
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The engrafted cells in each section that were positive for both Venus and each cell type-specific marker were counted (n = 4).
Nuclei were counterstained with DAPI and slides were coverslipped with paraphenylenediamine. Cell images were taken by Axiocam MRm camera system (Carl Zeiss Inc., Thornwood, NY, USA) coupled to a fluorescent microscope (Axiovert 200M, Carl Zeiss Inc .. Positive staining and total cells (2,000 cells for each marker) were counted and analyzed as described in the immunocytochemistry method.
Three brains per genotype per marker were counted.
Number of TILs that immunostained positively for each marker were counted using NIH ImageJ software.
To calculate recombination frequencies, the number of parental (AA and BB) and recombinant gametes (AB and BA) between a GBS marker and a reference marker were counted.
All the heterozygous cases, and those that were constitutionally homozygous (non-informative) for a marker, were counted to estimate the MSI rate.
Positive cells in the selected areas for each T-cell marker were counted independently by two investigators without knowledge of clinical information.
The adjusted positive predictive value (PPV*) is similar to the standard PPV, except that any selected nonzero coefficient falling within 500 kb of a GIANT-identified marker is counted as a true positive [ 39].
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