This video demonstrates the preparation, immunostaining, and imaging of neuromuscular junctions in rat muscle tissue using confocal and super-resolution microscopy to visualize and analyze presynaptic and postsynaptic structures.
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Method Article
Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy
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June 17th, 2025
In This Article
Abstract
Protocol
All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation
1. Tease each muscle in small fiber bundles of about 1 mm wide using two fine serrated forceps.
NOTE: It is crucial to manipulate muscles very gently with the forceps, without excessive force, to prevent tissue damage during teasing.
1. Dissociate the tibialis anterior (TA) muscle into 3 or 4 bundles depending on its size.
2. For gastrocnemius (GA), separate the medial and ....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Buffers and Reagents | |||
| Alexa Fluor 488 goat anti-mouse IgG (F488) | Life Technologies, Thermofisher | A-11001 | |
| Alexa Fluor 488 α-bungarotoxin (F488-a-BTX) | Life Technologies, Thermofisher | B13422 | |
| Alexa Fluor 594 goat anti-mouse IgG (F594) | Life Technologies, Thermofisher | A-11032 | |
| ATTO-633 α-bungarotoxin (F633-a-BTX) | Alomone Labs | B-100-FR | |
| Bovine serum albumin (BSA) | Sigma | A2153 | |
| DAPI Fluoromount-G | Southern Biotech | 00-4959-52 | |
| Dulbecco's Phosphate Buffered Saline (DPBS) | Gibco, Invitrogen | 14190-169 | |
| Ethanol Absolute | VWR | 20821.296 | |
| Triton X-100 | Sigma | T8787 | |
| Immersion Oil, n = 1.518 | THORLABS | MOIL-10LF | Low autofluorescence |
| Neurofilament (NF-M) antibody | DSHB | AB_531793 | |
| Paraformaldehyde (PFA) | MERCK | 1.04005 | |
| Synaptic vesicle glycoprotein 2 (SV2) antibody | DSHB | AB_2315387 | |
| Materials | |||
| Alnico Button cylindrical magnets | Farnell France | E822 | Diameter of 19.1 mm with maximal pull of 1.9 Kg |
| 63x 1.4 NA magnitude Oil immersion HCX Plan Apo CS objective | Leica Microsystems | ||
| 100x 1.4 NA HC PL APPO CS2 Objective | Zeiss | ||
| Fine serrated forceps | Euronexia | P-95-AA | |
| Curved thin forceps-Moria iris forceps | Fine Science Tools | 11370-31 | |
| Extra thin scissors - Vannas-Tübingen Spring Scissors | Fine Science Tools | 15-003-08 | |
| Gel loading tip round 1-200 μL | COSTAR | 4853 | |
| Leica laser-scanning confocal microscope TCS SP8 | Leica Microsystems | ||
| Leica Laser-scanning confocal microscope TCS SP8 Gated STED775 nm | Leica Microsystems | ||
| Lens Cleaning Tissue | Whatman (GE Healthcare) | 2105-841 | |
| Superfrost plus slides | Thermo Scientific | J1800AMNZ | |
| Medium serrated forceps | Euronexia | P-95-AB | |
| Microscope cover glasses 24x50 nm No 1.5H 170±5 µm | Marienfield | 107222 | High precision |
| Nunclon delta surface (12-well plates) | Thermo Scientific | 150628 | |
| Nunclon delta surface (24-well plates) | Thermo Scientific | 142475 | |
| Software | |||
| GraphPad | Prism, San Diego (US) | Release N°6.07 | Statistical software |
| ImageJ software | National Institutes of Health | Release N° 1.53f | |
| Leica Application Suite X software | Leica Microsystems | Release N°3.7.2.2283 | Free microscope softwareavailable at https://www.leicamicrosystems.com/products/microscope-software/p/leica-las-x-ls/downloads/ |
