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Since fluorescence intensity was quantified for each image, the gain and offset settings of the microscope were kept constant over the duration of each experiment.
Mann-Whitney U test was used for statistical analysis Care was taken to stain control and β2−/−/γ3−/− cells in parallel using the same solutions as well as to acquire the confocal images with exactly the same settings of the microscope.
Keeping the settings of the microscope constant, images were captured.
Controls with incubations of solitary secondary antibodies were carried out routinely with identical settings of the microscope.
All optical settings of the microscope were kept constant for all image acquisitions, in order to be able to compare the acquired information.
Following staining, embryos were cleared in benzyl benzoate/benzyl alcohol (2 1 vol/vol) Five random embryos were chosen for each probe dose combination and imaged from the animal cap in one session with no changes to the settings of the microscope.
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For the quantitative analysis of Smo levels in cilia, all images used for comparisons were taken with identical gain, offset, and laser power settings on the microscope.
Optimal edge contrast is achieved by adjusting the settings on the microscope and the camera.
The laser, pinhole and gain settings of the confocal microscope were kept identical among different treatments.
For semi-quantitative measurement of fluorescence intensities, laser, pinhole and gain settings of the confocal microscope were kept identical among treatments.
The settings of the multiphoton microscope were as previously described.
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