Synapsin-1 was localized in the levator auris longus (LAL) muscle by immunohistochemistry (IHC) in accordance with the protocol presented in Cilleros-Mañé et al. [41 (link)]. LAL muscles from the same animals that we used for western blot were used to perform this technique (n = 3).
As a control, primary antibodies were omitted from some muscles during the immunohistochemical procedures. These control muscles never exhibited positive staining. In double-staining protocols, omitting either one of the two primary antibodies completely abolished the corresponding staining and there was no cross-reaction with the other primary antibody. At least three muscles were used as negative controls.
A laser-scanning confocal microscope (Nikon TE2000-E) was used to study immunolabeled NMJs from the whole-mount muscles [41 (link)]. FIJI (ImageJ) software was used to perform 3D colocalization analyses from confocal stacks. The Pearson correlation coefficient (r) was used for quantitative analysis of colocalization. This statistic coefficient provides the overall association of two probes in an image. Images were assembled using Adobe Photoshop software (Adobe Systems, San Jose, CA), and neither the contrast nor brightness was modified.
As a control, primary antibodies were omitted from some muscles during the immunohistochemical procedures. These control muscles never exhibited positive staining. In double-staining protocols, omitting either one of the two primary antibodies completely abolished the corresponding staining and there was no cross-reaction with the other primary antibody. At least three muscles were used as negative controls.
A laser-scanning confocal microscope (Nikon TE2000-E) was used to study immunolabeled NMJs from the whole-mount muscles [41 (link)]. FIJI (ImageJ) software was used to perform 3D colocalization analyses from confocal stacks. The Pearson correlation coefficient (r) was used for quantitative analysis of colocalization. This statistic coefficient provides the overall association of two probes in an image. Images were assembled using Adobe Photoshop software (Adobe Systems, San Jose, CA), and neither the contrast nor brightness was modified.
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