One‐Step Vital Staining of Presynaptic Terminals and Post‐Synaptic Receptors at Neuromuscular Junctions in Mouse Skeletal Muscle

Richard R. Ribchester1

1 Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, Scotland, United Kingdom
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110128
Online Posting Date:  January, 2014
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Abstract

This protocol describes vital staining of neuromuscular junctions in the mouse triangularis sterni muscle in one incubation step, combining presynaptic, motor nerve terminal staining with the styryl dye FM1‐43, which labels recycling synaptic vesicles, and TRITC‐α‐bungarotoxin, which labels acetylcholine receptors in the motor endplate membrane. Curr. Protoc. Mouse Biol. 1:489‐496 © 2011 by John Wiley & Sons, Inc.

Keywords: neuromuscular junction; fluorescence microscopy; vital staining; styryl dyes; α‐bungarotoxin; acetylcholine receptor; synaptic vesicle

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Physiological saline (see recipe)
  • Depolarizing saline (see recipe)
  • Adult mice (any laboratory strain, e.g., C57B16; any age or gender)
  • 1 mg/ml FM1‐43 or FM1‐43FX (see recipe)
  • 500 µg/ml TRITC‐α‐bungarotoxin stock solution (see recipe)
  • 4% paraformaldehyde, optional
  • Minutien pins
  • Sylgard‐lined petri dishes
  • Dissecting microscope fitted with lamps or fiber‐optic illumination for both transmitted and incident light
  • Watchmaker's forceps (nos. 3 and 5)
  • Fine spring scissors
  • Iris scissors
  • Incubator with rocking platform
  • Upright fluorescence microscope or confocal microscope fitted with water‐dipping objectives
  • Digital camera and driver/image‐processing software/PC
  • Additional reagents and equipment for euthanizing the animal (Donovan and Brown, )
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Figures

Videos

Literature Cited

Literature Cited
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