Seeking better treatments for pretermbabies in the enteric nervous system NatureComms
) for 2 h at room temperature while shaking, washed twice in PBT, treated with PBS containing DAPI for 5 min, and washed twice in PBS. For mounting, tissue was submerged in ddHO, gently placed onto slides using paintbrushes under a dissection microscope, and allowed to briefly air dry before being coverslipped with Fluoromount-G .Images were captured on a Leica SP8 confocal microscope using a 20× oil objective.
To count neuronal subtypes as an overall percentage of HuC/D+ neurons, image stacks of HuC/D immunolabeling in MP wholemount preparations were blurred and thresholded. Because HuC/D+ neurons in our tissue were tightly packed and not easily resolved, we approximated neuronal number from the overall area of HuC/D labeling. The HuC/D stack was maximally projected, and the Analyze Particles function was used to determine the total area labeled with HuC/D.
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