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Protocol publication

[…] Image processing for ciliary SMO levels was carried out using maximum projection images of the acquired Z-stacks using ImageJ. For quantification of ciliary Smo, first a mask was constructed using the Arl13b image (primary cilia marker), and then the mask was applied to the corresponding Smo image where the integrated fluorescence was measured. An identical region outside the cilia was measured to determine background fluorescence. Background correction was applied on a per cilia basis by subtracting the background fluorescence from the cilia fluorescence.For neural differentiation experiments, fluorescent images were collected on a Leica TCS SP8 confocal imaging system equipped with a 40x oil immersion objective using the Leica Application Suite X (LASX) software. For each experiment, coverslips from each condition were grown, collected, and processed together to ensure that the cells were fixed and stained for the same duration of time. To ensure uniformity in imaging, the gain, offset, and laser power settings on the microscope were held constant for each antibody. At least 15 images were taken per condition. To ensure all cells were represented, z-stacks were acquired and counts were performed on the compressed images. Cell counts were conducted using the NIH ImageJ software suite with cell counter plugin. In total, 5000–6000 cells were analyzed per condition. The experiment was conducted independently a total of three times. Representative images shown in were processed equally using Adobe Photoshop, Adobe illustrator, and CorelDraw software. […]

Pipeline specifications

Software tools ImageJ, LAS X, Adobe Illustrator, CorelDraw
Applications Miscellaneous, Microscopic phenotype analysis
Chemicals Cholesterol, Cysteine