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Tissue Processing

Immunofluorescence staining and imaging

Human Muscle Tissue

Confocal image of a human muscle biopsy stained with antibodies to a-actinin (blue) at the Z line, the nebulin M1M2M3 domain (green) near thin filament pointed ends and rhodamine phalloidin for F-actin (from D. Gokhin, N. Kim, V. Fowler).

The SDMRC Imaging Core has developed and optimized tissue processing and immunostaining procedures for a diverse array of muscle tissue preparations, ranging from isolated myofibrils, to insect flight muscles, to cryosections of intact rodent muscles, all the way up to human muscle biopsies (1-5).  Tissue processing services provided by the Imaging Core include assistance with muscle tissue immobilization, relaxation, fixation, and (when appropriate) embedding and cryosectioning.  Tissue processing will be followed by blocking and treatment with primary antibodies for the investigator’s target proteins of interest as well as fluorescently labeled secondary antibodies.  The Imaging Core will provides detailed protocols for investigators wishing to perform tissue processing and immunostaining procedures in their own laboratories.  Finally, the Imaging Core is happy to provide its expertise and guidance with respect to selection of appropriate antibodies and imaging modalities to address the specific technical requirements imposed by the investigator’s particular scientific problem. 

 

Transmission electron microscopy

The SDMRC Imaging Core utilizes the TSRI Microscopy Core directed by Dr. Malcolm R. Wood, a leader in electron microscopy of biological samples.  As a result, the TSRI Microscopy Core has abundant expertise in electron microscopy of striated muscle tissues (1,3,6,7), and its services are available to those who wish to perform transmission electron microscopic analysis of muscle cells and tissue ultrastructure.  Electron microscopy services include tissue fixation (perfusion fixation for rodent tissues), embedding, sectioning, heavy metal staining, and imaging. The TSRI Microscopy Core is also adept at performing immunogold labeling followed by immunoelectron microscopy of tissues for ultra-high-resolution localization of target proteins.  Staff of the TSRI Microscopy Core is available for consultation to determine the optimal electron microscopy-based strategy for an investigator’s biological problem.

Mouse Muscle Tissue

TEM image of mouse TA muscle (from D. Gokhin, M. Wood, V. Fowler).

 

References

  1. Nowak, R. B., and Fowler, V.M. (2012) Tropomodulin 1 Constrains Fiber Cell Geometry during Elongation and Maturation in the Lens Cortex. JHC 60, 6, 414-427
  2. Yamashiro, S., Gokhin, D. S., Kimura, S., Nowak, R. B., and Fowler, V. M. (2012) Tropomodulins: Pointed-End Capping Proteins That Regulate Actin Filament Architecture in Diverse Cell Types. Cytoskeleton Apr 4,
  3. Gokhin, D. S., and Fowler, V. M. (2011) Cytoplasmic gamma-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers. J Cell Biol 194, 105-120
  4. Littlefield, R., and Fowler, V. M. (2002) Measurement of thin filament lengths by distributed deconvolution analysis of fluorescence images. Biophys J 82, 2548-2564
  5. Mardahl-Dumesnil, M., and Fowler, V. M. (2001) Thin filaments elongate from their pointed ends during myofibril assembly in Drosophila indirect flight muscle. J Cell Biol 155, 1043-1053
  6. Gokhin, D. S., Kim, N. E., Lewis, S. A., Hoenecke, H. R., D'Lima, D. D., and Fowler, V. M. (2012) Thin-filament length correlates with fiber type in human skeletal muscle. Am J Physiol Cell Physiol 302, C555-565
  7. Castillo, A., Nowak, R., Littlefield, K. P., Fowler, V. M., and Littlefield, R. S. (2009) A nebulin ruler does not dictate thin filament lengths. Biophys J 96, 1856-1865
  8. Conti, F.J., Felder, A., Monkley, S.J., Schwander, M., Wood, M.R., Lieber, R., Critchley, D., and Müller, U. (2008) Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle. Development 135, 2043-53
  9. Conti, F.J., Monkley, S.J., Wood, M.R., Critchley, D.R., and Müller, U. (2009) Talin 1 and 2 are required for myoblast fusion, sarcomere assembly and the maintenance of myotendinous junctions. Development 136, 3597-606