Journal: Scientific Reports
Article Title: R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5
Figure Lengend Snippet: Lack of R-spondin 2 (Rspo2) in mice has minimal effects on spinal motor neuron (SMN) survival and muscle differentiation, but has a significant effect on acetylcholine receptor (AChR) clusters in the left diaphragm. ( A ) Immunostaining for islet1/2 expressed in the SMNs of the spinal cord (C3-C6) at embryonic day ( E ) 18.5. ( B ) The number of islet1/2-positive SMNs in wild-type (+/+), heterozygous Rspo2 -knockout (+/−), and homozygous Rspo2 -knockout (−/−) mice. Bars indicate the mean and standard error of mean (SE) ( n > 90). No statistically significant differences (n.s.) were observed with one-way ANOVA. ( C,D ) Hematoxylin and eosin staining of the tibialis anterior muscle of mice at E18.5. ( E–H ) Representative electron micrographs of the diaphragms at E18.5 at different magnifications. ( I ) Thickness of muscle fibers at the Z disk in the left diaphragms of wild-type (+/+) and Rspo2 -knockout (−/−) mice. Five to seven electron micrographs were analyzed in each mouse. ( J ) Surface views of the left diaphragms harvested from wild-type (+/+) and Rspo2 -knockout (−/−) mice at E18.5. AChR was stained with Alexa546-conjugated α-bungarotoxin (red). The widths of the AChR bands of wild-type and Rspo2 -kockout diaphragms were 143.34 ± 3.73 μm and 221.85 ± 6.52 μm, respectively ( p
Article Snippet: After the removal of the connective tissue, the whole-mount diaphragm was permeabilized with 0.5% Triton X-100 in PBS for 10 min and then incubated overnight with α-bungarotoxin conjugated with the Biotin-XX Microscale Protein Labeling Kit (1:800, Invitrogen), anti-peripherin antibody (1:800, Millipore, AB1530), and anti-synaptophysin antibody (1:100, Invitrogen, 180130).
Techniques: Mouse Assay, Immunostaining, Knock-Out, Staining