Muscle Tissue Analyis

Muscle biopsies are routinely taken from the vastus lateralis muscle. First, an anesthetic (1% xylocaine) is injected locally in skin, soft tissue below, and the muscle fascia in the middle region of the vastus lateralis muscle. Thereafter, a small incision (3-5 mm) is made through the skin and the fascia at approx. 15 cm above the patella. An adapted Bergstrom needle is inserted to a depth of approx. 2–3 cm below the entry of the fascia and a muscle sample (between 60-120 mg) is generally obtained by suction. Muscle tissue is freed from any visible non-muscle material. Generally, one portion is immediately frozen in liquid nitrogen for biochemical analyses, another portion is frozen in nitrogen-cooled isopentane and embedded in Tissue-Tek for immunohistochemical analyses. Both samples are stored at -80oC until further analyses.



Biochemical analyses of muscle tissue
In our lab we are routinely processing small muscle tissue samples for biochemical assays. The latter varies from the measurement of muscle metabolite concentrations (like: ATP, ADP, AMP, creatine, creatinephosphate, glycogen, intramyocellular lipid, lactate, etc) to a large selection of enzyme activity assays (like: cytochrome C oxidase, succinate dehydrogenase, carnitine palmitoyl transferase, glycogen synthase, glutamate dehydrogenase). Besides mixed muscle samples, we are experienced in single fiber preparations, allowing muscle fiber type specific analyses. Furthermore, muscle tissue preparations for glucose, palmitate, and amino acid tracer enrichment analyses at the SIRC are also performed on a regular basis.


Histochemistry on muscle cross-sections
In our lab, standard histological and (immuno)histochemical techniques are routinely used on muscle tissue cross-section, to determine various anatomical, chemical and/or metabolic properties of the tissue under investigation. These techniques are generally being used for both cross-sectional comparisons of muscle characteristics between different groups but also for within subject variations in response to the short- and/or long-term effects of exercise and/or nutritional interventions.


The standard techniques being used include amongst others:
- Muscle fiber type composition based on both ATPase and MHC stainings
- SDH staining for determining SDH activity / oxidative capacity of muscle fibers
- Glut4 staining for determining Glut4 content and localization in muscle fibers
- ORO staining for determining intramyocellular lipid storage
- PAS staining for determining intramyocellular glycogen storage
- CD31 staining for determining microvascularisation of muscle tissue
- pax7/CD56 staining for determining myocellular satellite cell content


Generally, different stainings are also combined on either the same section or on serial muscle cross sections yielding additional information, e.g. on the fiber type specificity of certain metabolic properties (fiber type specific glycogen or lipid content) or myocellular characteristics (fiber type specific satellite cell content).



Example of fiber type specific analysis of satellite cell content. A: section of muscle tissue stained for MHC-I (green), laminin (red) and myonuclei (blue). B: serial cross-section from area depicted in the frame in A, but stained for laminin (red), myonuclei (blue) and satellite cells (green, depicted by the arrow). Type I and II muscle fibers are indicated.


The standard procedures and equipment being used throughout the process are:
-Sectioning of the tissue on a cryostat, usually cutting 5µm thick cross sections of muscle tissue and orienting the tissue for cross-sectional analyses of muscle fibers
-Staining of the tissue with enzyme or antibody (immuno) histochemistry
-Recording of the stained sections with a combined light / immunofluorescence microscope (Nikon E800) coupled to a Basler A113C progressive scan color CCD camera with a Bayer color filter. With this setup, epifluorescence signal is recorded using a Texas Red excitation filter (540–580 nm) for red labels, a FITC excitation filter (465–495 nm) for green labels, and a DAPI UV excitation filter (340–380 nm) for blue labels
-Image analyses using Lucia 4.81 software package (Nikon) for determining anatomical properties (e.g. muscle fiber size, composition), metabolic properties (e.g. intensity of ORO or PAS stainings) or combinations of both (i.e. specific localization of metabolic properties throughout the muscle fiber area, or layer-quantification, i.e. central vs subsarcolemmal).
For a more detailed description and some examples of analytical techniques, methods and applications in our research we refer to our publications:


mRNA and protein expression
For mRNA and protein expression analyses in skeletal muscle tissue collected in numerous human in vivo studies, we generally apply RT-PCR and Western blotting respectively. A few of the key-proteins we measure routinely in muscle tissue include: mTOR, S6K1, S6, AMPK, Akt, 4E-BP1, eEF2, GSK-3B, eIF4B, eIF2alpha, FAT/CD36, FABP, GLUT-4, etc. For these analyses we use systems like: the protean mini trans blot, criterion and II xi, the Chemidoc system and immunochemiluminiscent methods for detecting our proteins of interest. The protein content of the biopsies is generally measured by a Bradford assay. We also use Coomassie-, Ponceau-S- and Silver-staining methods for the detection of our proteins on blots or in acrylamide gels (self poured or precast systems). Gels or X-ray films are being scanned on the GS 800 calibrated densitometer. Image analyses is generally performed using the Quantity One 4.2.1 software package. For mRNA expression levels we are using the MyiQ single color real time PCR I-Cycler. For a more detailed description and some examples of analytical techniques, methods and their applications we refer to our publications.