Soleus muscles were dissected, pinned in mild stretch, and fixed by immersion for 20 minutes in 4% para-formaldehyde with phosphate-buffered saline (PBS; pH 7.4). After rinsing in PBS, muscles were soaked in 15% sucrose with PBS (overnight at 4°C), followed by 30% sucrose with PBS for at least 24 hours. Muscle tissues were embedded in OCT and sectioned with a cryostat (HM 550; Microm GmbH, Walldorf, Germany) into 40-mm sections and placed on glass slides for staining. For a pre-synaptic marker, sections were incubated with βIII-tubulin (1:500; Promega Catalog No. G7121) antibody overnight at 4°C, followed by secondary fluorescein isothiocyanate–labeled goat anti-mouse (1:100; Jackson ImmunoResearch Catalog No. 115-095-205) antibody. Rhodomine bungarotoxin (1:40, ThermoFisher Catalog No. B13423) was used as a postsynaptic marker. Stained sections were examined under a fluorescence and confocal microscope. The Zeiss LSM Image Examiner version 1.0.0.241 (Carl Zeiss, Oberkochen, Germany) was used for imaging analysis.
Fluorescein isothiocyanate labeled goat anti mouse
Manufactured by Jackson ImmunoResearch
Fluorescein isothiocyanate–labeled goat anti-mouse is a secondary antibody conjugate produced in goats and labeled with the fluorescent dye fluorescein isothiocyanate (FITC). This product is designed for use in immunoassays and other fluorescence-based applications.
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2 protocols using fluorescein isothiocyanate labeled goat anti mouse
1
Immunohistochemical Analysis of Neuromuscular Junctions
2
Histological Analysis of Neuromuscular Junctions
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Extensor digitorum longus (EDL) muscles were dissected, pinned in mild stretch, and fixed by immersion for 20 minutes in 4% paraformaldehyde with PBS (pH 7.4). After rinsing in PBS, muscles were soaked in 15% sucrose with PBS (overnight at 4°C), followed by 30% sucrose with PBS for at least 24 hours. Muscle tissues were embedded in OCT and sectioned with a cryostat (HM 550; Microm GmbH) into 40-mm sections and placed on glass slides for staining. For a presynaptic marker, sections were incubated with βIII-tubulin (1:500; Promega, catalog G7121) antibody overnight at 4°C, followed by secondary fluorescein isothiocyanate–labeled goat anti-mouse (1:100; Jackson ImmunoResearch, catalog 115-095-205) antibody. Rhodamine bungarotoxin (1:40, Thermo Fisher Scientific,, catalog B13423) was used as a postsynaptic marker. Stained sections were examined under fluorescence and confocal microscopy. The Zeiss LSM Image Examiner version 1.0.0.241 (Carl Zeiss) was used for imaging analysis. We quantitated pre- over postsynaptic NMJ areas, and this method has shown to be effective in detecting differences in NMJ connectivity in published studies (64 (link)).
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