The software Image Pro Plus, which is specialized in fluorescent cell counting, was applied to calculate the number of viable cells in each picture as the cell density at the intercross point.
SLAC (single likelihood ancestor counting) was applied to calculate the average nonsynonymous substitution/synonymous substitution rate (dN/dS) for the nine genes (Kosakovsky Pond and Frost 2005).
For determination of the spores number, the above serially diluted mixture was further heat-treated at 80 °C for 15 min to kill the vegetative cells in the diluted sample and then the same procedure for colony counting was applied [ 19].
TH-positive SN cells were counted by stereology blind with regard to the experimental condition using a Leica DM6000B motorized microscope coupled with the Mercator Pro software (ExploraNova, La Rochelle, France).The SN was delineated for each section and probes for stereological counting were applied to the map obtained (sampling and size of probes, see Additional file 4: Table S3).
Standard corrections for CT-based attenuation, scatter, decay, and random counts were applied.
Starting with a dataset of 149,291 unique transcript tags, a second filter selecting for a project total (sum expression level all 22 libraries included in this study) of ≥10 counts was applied reducing the amount of transcript tags to 23, 649.
To determine whether RNA is detected, a cutoff value of the normalized read count was applied.
Thresholds in force, distance, and peak counts were applied to sort out nonspecific and multiple interactions.
Data were cross-tabulated by age-group (27 to 50 years, 51 to 60 years, 61 to 70 years, and 71 years and over) and gender, and chi-squared or Fisher's exact tests (where there were less than 5 cell counts) were applied.
The raw camera images are transferred to a PC and an event counting method is applied directly after acquisition.
