Morphological analysis of neuromuscular junctions by immunofluorescent staining of whole-mount mouse diaphragms

Immunofluorescence or IF is a technique allowing the visualization of a specific protein or antigen in cells or tissues by binding a specific antibody chemically conjugated with a fluorescence dye. Immunofluorescent staining is widely used in life science research, particularly for neuroscience. Here, we describe the immunofluorescent staining of whole-mount neonatal mouse diaphragms to study the morphological patterns of the neuromuscular junction (NMJ) by using of presynaptic neuronal marker-neurofilament (NF) and synaptophysin antibodies; postsynaptic acetylcholine receptors (AChRs) were labeled with Alexa Fluor 594-conjugated α-bungarotoxin (α-BTX). Immunofluorescence-stained diaphragms were examined under a confocal microscope.