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Confocal micrograph measurements of mdx and WT mouse NMJs

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posted on 2018-07-20, 15:58 authored by Seth HaddixSeth Haddix

Confocal micrographs were collected on either a Leica TPS II SP5 or a Zeiss LSM780 microscope using a 40X Oil objective (NA 1.4). Step size was set between 300-500 nm. Laser lines 405 nm, 458 nm, 488 nm, 563 nm, and 633 nm were used as necessary. Images were analyzed in ImageJ (ImageJ, RRID:SCR_003070) by creating maximum projection images from collected stacks. Junctional Area was calculated by thresholding the fluorescent bungartoxin labeled channel and drawing a polygon around the object and measuring the shape’s area. Receptor Area was measured as the thresholded area of the BTX label within the circumscribed region of the AChR. Dispersion Index was calculated as Receptor Area/Junctional Area. The number of AChR fragments (fragmentation) was measured using the objects counter in ImageJ by setting the minimum area for counts as 5 μm2. tSC counts were made by counting only fluorescently labeled Schwann cells in proximity to the endplate having coincident nuclear label via DAPI. Axon branch points were measured by creating 3D rotations of the confocal images of fluorescently labeled motor neurons, and then counting the number of times the axon split. Small varicosities of the motor terminal were not counted as branches.


Included is a compressed oib file of confocal collection. Best opened in ImageJ

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