This website uses cookies so that we can provide you with the best user experience possible. Cookie information is stored in your browser and performs functions such as recognising you when you return to our website and helping our team to understand which sections of the website you find most interesting and useful.
An ImageJ macro for the quantification of ciliary accessible cholesterol
Output Details
Description
ALOD4, a toxin-based probe, can be used to visualize the accessible pool of cholesterol (PMID: 30790255). Here we describe an algorithm for segmenting fluorescently-labeled cilia using SSTR3 as a ciliary marker. The segmented masks are used to determine the intensity of ALOD4-bound accessible cholesterol in the ciliary membrane. The algorithm asks for a directory path and searches for .tif-files. Each identified .tif-file is cropped for a user defined region of interest (ROI) which must contain the cilia in the image series. Then the Z-stack is maximum intensity projected, the contrast of channel 1 (SSTR3-mApple, red) is optimized for visualization, and both channels are duplicated. The MorphoLibJ library helps to identify the SSTR3-positive cilia. SSTR3 signals are then thresholded, binarized, and intensity-corrected. A shape filter prevents false positive signals by preventing non-ciliary structures to be assigned as objects. The identified objects are applied onto the channel 2 signal (mNeon-ALOD4, green) and intensities are measured and saved.
Identifier (DOI)
10.17504/protocols.io.q26g71d88gwz/v1