To validate the automated nuclear shape analysis, we also assessed nuclear morphology using the standard technique: manual blind counting.
To judge the impact of rapamycin and RAD001, we first scored the percentage of nuclei with abnormal morphology in the usual way by manual blind counting.
At least 500 cells per slide and three slides per condition were double-blind counted.
This approximation requires, besides confocal microscopy, the participation of a qualified and blind counting supervisor to avoid subjective appreciations of the imaging interpretation of the data.
Two people completed triple blind counts of each of the specimens and then the average of these counts was used as the age of the animal.
Cell migration was quantified by blind counting of the migrated cells on the lower surface of the membrane of 5 fields per chamber under microscope.
Accuracy of puncta quantification was verified by independent blind counting.
Our analysis shows that treatment with RAD001 and rapamycin reduces nuclear blebbing, consistent with blind counting controls.
The BrdU incorporation index was obtained by a blind counting of stained and unstained germ cell nuclei in all sections.
However, the extent of blebbing varies considerably within each cell population, which makes manual blind counting challenging and subjective.
