Hindlimb Skeletal Muscle Extraction: A Procedure to Excise Hindlimb Skeletal Muscles from Murine Model
JoVE Encyclopedia of Experiments
Cancer Research
0 views • 3:40 min • April 30th, 2023
To begin, prep a euthanized mouse in the supine position. Clamp one of its feet to extend the limb vertically. Gently hold the skin of the lower limb.
Next, surgically remove the skin and fascia, the layer of loose connective tissue just underneath the skin, exposing the underlying muscle bellies of the lower limb.
Identify the gastrocnemius muscle - a large, superficial two-headed muscle - on the posterior lower limb. It originates from the lateral and medial condyles of the posterior femur and attaches to the calcaneus - heel bone - via the calcaneal tendon.
Hold the distal end of the muscle and pull the muscle belly towards its origin. Excise the gastrocnemius calcaneal tendon followed by the muscle at the origin closest to the femur.
Now, locate the tibialis anterior muscle. The muscle originates from the anterolateral surface of the tibia and attaches to the medial cuneiform bone via the distal tendon.
Detach the tibialis muscle belly from the underlying connective tissue. Cut the distal tendon and the muscle at the origin closest to the tibia.
Finally, transfer the excised muscles into cryotubes. Store the tubes at low temperatures until further downstream analysis.
To avoid fur contamination, spray 70% ethanol over the entire body. Then, place the animal on a dissection bed in the supine position and attach one hind limb to an elevated buret clamp to extend the limb vertically. Using Dumont forceps, grasp the superficial skin of the hind limb, and use curved fine scissors to gently remove the skin and fascia, exposing the underlying muscle bellies of the lower limb.
Identify the gastrocnemius muscle on the posterior lower limb by its origin at the lateral and medial condyles on the posterior femur and its insertion at the calcaneus via the calcaneal tendon. Use the forceps to grasp the distal end of the gastrocnemius and pull the muscle belly toward its origin. Then, use scissors to cut the muscle at the origin as close as possible to the femur and transfer the muscle to a pre-weighed dish.
Weigh the dish immediately and transfer the muscle into a pre-labeled cryotube. Next, identify the tibialis anterior muscle from its origin at the anterolateral surface of the tibia and its insertion at the medial cuneiform via its distal tendon. Then, grasp the foot by the digits with the index finger and thumb and insert the tip of the Dumont forceps immediately under the superficial distal tendon of the tibialis.
Move the forceps such that the blunt side can be used to detach the tibialis muscle belly from the underlying connective tissue and use the fine curved scissors to cut the distal tendon. Then, cut the muscle at the origin as close as possible to the tibia.
Overview
This article outlines a surgical procedure for isolating the gastrocnemius and tibialis anterior muscles from a euthanized mouse. The method includes detailed steps for muscle identification, excision, and preservation for further analysis.
Key Study Components
Area of Science
- Neuroscience
- Muscle Physiology
- Animal Surgery
Background
- The gastrocnemius and tibialis anterior muscles are crucial for studying muscle function and pathology.
- Understanding muscle anatomy is essential for various experimental applications.
- Proper dissection techniques are vital for preserving muscle integrity.
- Muscle samples are often used in metabolic and physiological studies.
Purpose of Study
- To demonstrate the surgical technique for muscle isolation in mice.
- To provide a protocol for subsequent analysis of muscle properties.
- To enhance understanding of muscle anatomy and its relevance in research.
Methods Used
- Preparation of the euthanized mouse in a supine position.
- Surgical removal of skin and fascia to expose muscles.
- Identification and excision of the gastrocnemius and tibialis anterior muscles.
- Storage of excised muscles in cryotubes for analysis.
Main Results
- Successful isolation of the gastrocnemius and tibialis anterior muscles.
- Preservation of muscle samples for downstream analysis.
- Detailed procedural steps ensure reproducibility in muscle dissection.
- Clear identification of muscle anatomy aids in experimental design.
Conclusions
- The described method provides a reliable approach for muscle isolation.
- Proper technique is crucial for maintaining sample integrity.
- This protocol can be adapted for various experimental needs in muscle research.
Frequently Asked Questions
Executive Industry Relevance
Precise isolation of skeletal muscle tissue enables reliable downstream biochemical and histological analysis in preclinical studies. This procedure supports target validation by providing high-integrity tissue samples for mechanistic de-risking of muscle-related pathways. Reproducible excision techniques enhance assay readiness and data consistency across discovery workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through functional analysis of excised muscle tissue.
- Operational Value: Provides standardized tissue preparation for consistent experimental outcomes.
- Predictive Value: Supports biological de-risking by preserving muscle integrity for downstream target engagement studies.
Screening & Assay Development
- Assay Readiness: Excised muscles serve as validated biological systems for compound screening and pathway modulation studies.
- Quantitative Output: Enables consistent measurement of muscle mass, protein content, and enzymatic activity.
- Reproducibility: Standardized dissection protocol reduces variability in preclinical assay results.
Translational & Preclinical Research
- Disease Relevance: Supports studies in muscle wasting models such as cancer cachexia and neuromuscular disorders.
- Translational Continuity: Provides tissue for biomarker analysis linking discovery to preclinical validation.
- Risk-Adjusted Advancement: Reliable tissue samples improve confidence in go/no-go decisions for muscle-targeted therapeutics.
Pipeline & Workflow Integration
This method fits within the discovery continuum from target identification to preclinical validation by enabling reliable tissue procurement for functional assays.
- Discovery Biology: Supports hypothesis testing and pathway clarification through isolated muscle tissue analysis.
- Screening: Prepares tissue for assay readiness in compound screening and target engagement studies.
- Analytics: Enables quantitative measurements such as muscle weight and protein yield for comparative analysis.
- Translational Research: Connects early discovery to preclinical validation via consistent tissue sampling for biomarker alignment.
- Enterprise Reuse: Establishes a reusable dissection capability across multiple muscle physiology and pathology projects.
Operational & Enterprise Impact
- Scientific Value: Enhances predictive confidence by preserving tissue integrity for downstream analysis.
- Operational Value: Ensures standardization and reproducibility in muscle tissue preparation.
- Strategic Value: Improves capital efficiency by reducing variability in preclinical outcomes.
- Portfolio Impact: Supports risk-adjusted prioritization through reliable tissue-based data.
Implementation Considerations
- Requires expertise in rodent anatomy and surgical dissection techniques.
- Necessitates sterile instruments and cryogenic storage infrastructure.
- Demands cross-team standardization for consistent tissue handling across sites.
- Involves adaptation considerations for different muscle groups and species.
- Limited by tissue degradation if not stored promptly at low temperatures.
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Last updated: 22 August 2026