We serially imaged the nerve grafts at the time of surgery and at 7, 14, 21, and 28 days later using a fluorescence dissecting microscope (MZ16FA Leica microsystem). To facilitate live imaging, mice were re-anesthetized and the grafted sciatic nerve was re-exposed and imaged with the fluorescent dissecting microscope for the exploration of SC migration and axonal elongation. To evaluate the distance of SC migration more accurately, SC migration from ETS coaptation was quantitatively assessed by preparing whole mount ANGs at 28 days after surgery. GFP-positive migrating cells in the entire graft could be observed using stack images from confocal microscopy.
The sciatic nerve graft was imaged with DFC300FX controlled by Leica Application Suite Advanced Fluorescence version 2.5 software (Leica microsystem) under GFP (488 nm) fluorescent and bright field filters. And the whole mount sciatic nerve graft was surveyed using TCS-SP5 (Leica microsystem). The images were recorded monochromatically using Leica Application Suite Advanced Fluorescence version 2.5 software (Leica microsystem). Images were standardized according to magnification (10×, 40×), exposure time (100–300 ms), orientation, brightness, and contrast.
The sciatic nerve graft was imaged with DFC300FX controlled by Leica Application Suite Advanced Fluorescence version 2.5 software (Leica microsystem) under GFP (488 nm) fluorescent and bright field filters. And the whole mount sciatic nerve graft was surveyed using TCS-SP5 (Leica microsystem). The images were recorded monochromatically using Leica Application Suite Advanced Fluorescence version 2.5 software (Leica microsystem). Images were standardized according to magnification (10×, 40×), exposure time (100–300 ms), orientation, brightness, and contrast.