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This was verified using a statistical colocalization analysis of confocal images (Fig. 3C and 3D).
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Statistical analysis was performed using a statistical analysis software [56].
Image analysis for NMJ analysis was performed using National Institutes of Health ImageJ software, and quantitative analysis of the NMJ morphology was performed using a colocalization analysis plugin.
A Zeiss 700 confocal microscope was used to image RNAscope for colocalization using a DIC filter to capture the nrg1 signal.
Colocalization was revealed using a scanning sequential mode to eliminate possible bleed-through effect.
Multi-color TIRF imaging was performed and the extent of colocalization was determined using a custom particle-tracking program (see 'Materials and methods').
The extent of a possible bi-peptide colocalization was measured fluorometrically using a 5-carboxyfluorescein (5-FAM)/QSY-7 fluorescence resonance energy transfer (FRET).
Colocalization was analyzed by using a confocal laser scanning microscope (LSM510; Zeiss, Tokyo, Japan).
The sections were photographed using a fluorescence microscope (Zeiss Axioskop, Carl Zeiss AG, Oberkochen) and assessed for pSTAT3/eGFP colocalization.
All colocalization quantifications were done using a Leica DMIRE2 epifluorescence microscope equipped with a 100× oil objective, 1.4 numerical aperture.
For colocalization studies, images were captured using a 60x objective (NA 1.45) on an Olympus FV-1000 confocal microscope at 5× digital zoom.
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