Journal: bioRxiv

Article Title: Genetic Rescue of a Lethal Wasting Mutation ( Syb1 lew/lew ) by Neuron-Specific Expression of ECFP-Syb2

doi: 10.64898/2026.05.22.727260

Figure Lengend Snippet: A: Transverse sections of lumbar spinal cord from a 4-month-old wild-type mouse were double labeled with antibodies against Syb1 (red) and Syb2 (green). Syb1 is enriched in the ventral horn, whereas Syb2 is more prominent in the dorsal horn. B: High-magnification images of the ventral horn region shown in A, highlighting a motor neuron (MN) and surrounding nerve terminals. Both Syb1 and Syb2 are detected in presynaptic terminals to the motor neuron. Syb1 and Syb2 are co-localized in a subset of terminals (arrowheads), with partial co-localization alongside terminals expressing either isoform alone. C, D: Whole-mount lumbrical muscles from 4-month-old wild-type mice were double-labeled with Texas Red-conjugated α-bgt and antibodies against Syb1( C ) or Syb2 ( D ). Both Syb1 and Syb2 are detected at all motor nerve terminals of the NMJs.

Article Snippet: Briefly, lumbrical or triangularis sterni muscles were fixed in 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) overnight at 4 ° C. Muscle samples were then incubated with Texas-Red conjugated α-bungarotoxin (α-bgt) (2 nM, Invitrogen, Carlsbad, California, USA) for 30 minutes, followed by incubation with primary antibodies diluted in antibody dilution buffer (500 mM NaCl, 0.01 M phosphate buffer, 3% BSA and 0.01% thimerosal) overnight at 4 ° C. The following primary antibodies were used: rabbit polyclonal anti-synaptotagmin2 (I735) , rabbit polyclonal anti-synaptobrevin1 (P938) and rabbit polyclonal anti-synaptobrevin2 (P939) (generous gifts from Dr. Thomas Südhof, Stanford University School of Medicine, Palo Alto, CA, USA).

Techniques: Labeling, Expressing, Muscles

Journal: bioRxiv

Article Title: Genetic Rescue of a Lethal Wasting Mutation ( Syb1 lew/lew ) by Neuron-Specific Expression of ECFP-Syb2

doi: 10.64898/2026.05.22.727260

Figure Lengend Snippet: A: Transverse section of lumbar spinal cord from a 5-month-old ECFP-Syb2 mouse labeled with TO-PRO-3 to visualize nuclei. CFP fluorescence is prominently detected in the gray matter. B: High-magnification image of a motor neuron in the ventral horn. CFP fluorescence is enriched in nerve terminals (arrows) contacting the motor neuron. The arrowhead indicates the nucleus of the motor neuron. C: Whole mounts of triangularis sterni muscles from 5-month-old wild-type and ECFP-Syb2 mice were double-labeled with Texas Red-conjugated α-bungarotoxin (α-bgt) to label AChRs and antibodies against the presynaptic marker synaptotagmin 2 (Syt2) to label motor nerve terminals. ECFP-Syb2 is present at all NMJs and overlaps with the presynaptic marker Syt2. D: High-magnification images show that ECFP-Syb2 co-localizes with Syt2 at presynaptic terminals and is juxtaposed to AChR clusters, with NMJ morphology comparable to wild-type mice.

Article Snippet: Briefly, lumbrical or triangularis sterni muscles were fixed in 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) overnight at 4 ° C. Muscle samples were then incubated with Texas-Red conjugated α-bungarotoxin (α-bgt) (2 nM, Invitrogen, Carlsbad, California, USA) for 30 minutes, followed by incubation with primary antibodies diluted in antibody dilution buffer (500 mM NaCl, 0.01 M phosphate buffer, 3% BSA and 0.01% thimerosal) overnight at 4 ° C. The following primary antibodies were used: rabbit polyclonal anti-synaptotagmin2 (I735) , rabbit polyclonal anti-synaptobrevin1 (P938) and rabbit polyclonal anti-synaptobrevin2 (P939) (generous gifts from Dr. Thomas Südhof, Stanford University School of Medicine, Palo Alto, CA, USA).

Techniques: Labeling, Fluorescence, Muscles, Marker

Journal: bioRxiv

Article Title: Genetic Rescue of a Lethal Wasting Mutation ( Syb1 lew/lew ) by Neuron-Specific Expression of ECFP-Syb2

doi: 10.64898/2026.05.22.727260

Figure Lengend Snippet: A : Whole mounts of triangularis sterni muscles from wild-type, Syb1 lew/lew and Syb1 lew/lew ;ECFP-Syb2 mice at P14 were double-stained with Texas Red-conjugated α-bgt and antibodies against Syb1. Syb1 is detected at wild-type NMJs but absent from either Syb1 lew/lew or Syb1 lew/lew ;ECFP-Syb2 NMJs. ECFP-Syb2 is detected at NMJs of Syb1 lew/lew ;ECFP-Syb2 mice. B : Whole mounts of triangularis sterni muscles from wild-type and Syb1 lew/lew ;GFP--Syb2 mice at 2 months of age. NMJ morphology in S yb1 lew/lew ;ECFP-Syb2 mice is comparable to that of wild-type NMJs.

Article Snippet: Briefly, lumbrical or triangularis sterni muscles were fixed in 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) overnight at 4 ° C. Muscle samples were then incubated with Texas-Red conjugated α-bungarotoxin (α-bgt) (2 nM, Invitrogen, Carlsbad, California, USA) for 30 minutes, followed by incubation with primary antibodies diluted in antibody dilution buffer (500 mM NaCl, 0.01 M phosphate buffer, 3% BSA and 0.01% thimerosal) overnight at 4 ° C. The following primary antibodies were used: rabbit polyclonal anti-synaptotagmin2 (I735) , rabbit polyclonal anti-synaptobrevin1 (P938) and rabbit polyclonal anti-synaptobrevin2 (P939) (generous gifts from Dr. Thomas Südhof, Stanford University School of Medicine, Palo Alto, CA, USA).

Techniques: Muscles, Staining