BIOQUANT OSTEO
Muscle Phenotyping Protocol


Definition of Muscle Phenotyping

Muscle phenotyping is the quantitative characterization of muscle cells to assess the practical impact of a particular genotype on muscle function. Common analytical end points include: the cross-sectional area of myocytes, the number of myocytes that express different myosin heavy chain isoforms, the distribution of myocytes of different types within the whole muscle, the number of satellite cells associated with each myocyte, and the distribution of satellite cells relative to their associated myocyte.

Staining

Many different staining techniques are used in the analysis of muscle tissue. The preferred method for a project depends on the type of data that must be collected.

ImmunofluorescenCE for morphology

Immunofluorescent staining. Red: laminin. Green: dystrophin. Blue: satellite cells.
Multi-Parametric MRI at 14T for Muscular Dystrophy Mice Treated with AAV Vector-Mediated Gene Therapy
(
http://dx.doi.org/10.1371/journal.pone.0124914.g005).

Ideally, identification of the borders of individual myocytes can be accomplished by immunostaining of dystrophin or laminin. This can be combined with nuclear staining to visualize satellite cells. Since myocytes frequently appear bundled together, having a specific label to indicate the boundary of each cell greatly increases the automation of cross-sectional area analysis.

Hematoxylin and eosin for morphology

Hematoxylin and eosin staining. Pink: myocytes. Purple: satellite cells.
Effects on Contralateral Muscles after Unilateral Electrical Muscle Stimulation and Exercise
(
http://dx.doi.org/10.1371/journal.pone.0052230).

Routine H&E staining, while fast and inexpensive, lacks the specificity needed for automated cross-sectional area analysis. Manual editing of the boundaries between myocytes will be needed.

ATPase Staining for Myocyte ISOFORM Typing

Alkaline Myosin ATPase Staining. Sections are paired top to bottom: A-E, B-F, C-G, D-H.
Unilateral Muscle Overuse Causes Bilateral Changes in Muscle Fiber Composition and Vascular Supply
(http://dx.doi.org/10.1371/journal.pone.0116455).

ATPase staining performed on serial tissue sections can be used to identify fiber types. Variations in pH control stain specificity to different heavy-chain isoforms. ATPase staining has the benefit of being relatively straightforward and inexpensive but the drawback of requiring multiple serial sections, one for each MyHC type visualized.

Multi-channel Fluorescence for Myocyte Isoforms

Multi-channel fluorescent staining. Red: type IIB. Blue: type I. Green: type IIA. Purple: IIX / IIAX.
Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis
(http://dx.doi.org/10.1371/journal.pone.0035273).

While more expensive and technically complex to perform, multi-channel immunofluorescence requires only a single section physical section and significantly simplifies imaging and automated fiber typing.

Imaging

Click image to enlarge.

Scanning of the section using either a digital pathology core facility or one of the BIOQUANT microscope scanning upgrades is recommended. This preserves the staining of the section and simplifies subsequent analysis. Scanning at 20X is typical, but 40X may be necessary depending on the pathology of the sample.

Muscle Boundary

Irregular Region of Interest

At low magnification, the boundary of the muscle is traced using the irregular ROI (region of interest) tool. The software uses this to calculate the area of the muscle and also to ensure cells are counted only with the sample area when working at higher magnification.

Detecting Satellite Cells

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In this section, the light green cells are the satellite cells. The cells are clumped enough and the stain color varies enough that we'll use manual thresholding instead of automatic. Using the Draw Threshold tool, place a representative dot over each satellite cell. The size of the dot is not important since only number and position data are required.

Once marked, BIOQUANT then automatically counts the satellite cells, records there x,y position, and plots them on a map of the section.

Detecting Positively Stained Myocytes

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Measure Cells Tool

Automated analysis of myocytes is possible, provided there is sufficient specificity in the staining. BIOQUANT learns the range of colors associated with the strongly stained myocytes and displays a threshold over the selected cells.

Additionally, BIOQUANT automatically excludes myocytes outside of the previously defined muscle area and myocytes that are only partially within the field of view (highlighted in pink).

Manual brush and eraser tools are available to edit the the threshold to correct any mistakes in the automatic detection of myocytes.

Once the myocytes are properly detected, BIOQUANT automatically quantifies cross-sectional area, location, and stain intensity.

Detecting Negatively Stained Myocytes

Click image to enlarge.

Measure Cells Tool

For the lightly stained myocytes, a similar procedure is used. The range of color is defined, manual edits are made as necessary to the threshold. Measure Cells ensure that no cell is counted twice and that only complete cells are counted. Green cells are measured. Pink cells are excluded since they are incomplete in the current field of view.

Repeat for Remaining Fields of View

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Automatic Region of Interest

Using the Large Image Navigator to move sequentially through the section, BIOQUANT automatically updates the region of interest so that no cells can be measured outside of the muscle area. In the above image the pink cells are selected for measurement. Note that darkly stained cells outside the green boundary of the muscle are ignored.

Skeletal Phenotyping Data

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BIOQUANT uses an inherently open data structure. This is a partial list of data that can be collected and computed for skeletal muscle phenotyping. New parameters are easily added.

Whole Muscle Data

  • Total Muscle Cross-sectional Area

Satellite Cell Data

  • Total Number of Satellite Cells
  • Mean Number of Satellite Cells per Myocyte
  • Position of Each Satellite Cell

Aggregate Myocyte Data

  • Total Number of Myocytes
  • Cross-sectional Area of Each Myocyte
  • Diameter of Each Myocyte
  • Stain Intensity (Optical Density) of Each Myocyte
  • Position of Each Myocyte

Positive Myocyte Data

  • Total Number of Positive Myocytes
  • Cross-sectional Area of Each Positive Myocyte
  • Mean Cross-sectional Area of Positive Myocytes
  • Diameter of Each Positive Myocyte
  • Mean Diameter of Positive Myocytes
  • Stain Intensity (Optical Density) of Each Positive Myocyte
  • Mean Stain Intensity (Optical Density) of Positive Myocytes
  • Position of Each Positive Myocyte
  • Ratio of Total Positive Myocyte Area / Total Muscle Area

Negative Myocyte Data

  • Total Number of Negative Myocytes
  • Cross-sectional Area of Each Negative Myocyte
  • Mean Cross-sectional Area of Negative Myocytes
  • Diameter of Each Negative Myocyte
  • Mean Diameter of Negative Myocytes
  • Stain Intensity (Optical Density) of Each Negative Myocyte
  • Mean Stain Intenstiy (Optical Density) of Negative Myocytes
  • Position of Each Negative Myocyte
  • Ratio of Total Negative Myocyte Area / Total Muscle Area