Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.
The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.
1. Preparation
2. Fixing Flies in Collars
Note: If you are analyzing several genotypes, do not forget to make a note of the collar number and corresponding genotype.
3. Paraffin Sections of Drosophila Thoraxes
4. Cryosections of Drosophila Thoraxes
5. Lipids Detection in Drosophila Muscles
Lipid droplets can be detected with oil red O stain on cryosections using a protocol adopted from Sieber and Thummel 5.
6. Representative Results:
Figure 1. Parrafin-embedded sections
Hematoxyline and eosine stained transverse (A-B) and longitudinal (C-D) sections of indirect flight muscles. A and C shows normal structured muscles. Abnormal by size and morphology muscles are represented on B and D respectively (black arrows). Transverse section of Drosophila thorax stained with anti-LamC, nuclear envelope marker and DAPI, nuclear stain (E-F). Enlarged view of normal (red arrow) and deteriorated (yellow arrow) muscles (F). G represents section of Drosophila intestinal tract stained with LamC and DAPI.
Figure 2. Frozen sections
A. Transverse frozen sections of Drosophila thorax stained with oil red O, lipid droplets label.
B. Longitudinal frozen sections of indirect flight muscles stained with anti-Dg, muscle sarcolemma marker and DAPI.
The authors have nothing to disclose.
We thank Prof. Eichele for allowing us to use the cryo-microtome. Work was funded by Max-Planck-Gesselschaft.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Stainless steel collars | self-made | Specially constructed | ||
Forceps | Fine Science Tools GmbH | 11295-10 | ||
Aluminum foil | Any | |||
Blade or scalpel | Any | |||
Wheaton macro staining jar | Wheaton Science Products | 900200 | ||
Microtome | Carl Zeiss | Model: Hyrax M25 | ||
Cryo-microtome | Leica | Model CM3050S | ||
60-65°C Incubator | Any | Large enough to hold at least 4 staining jars | ||
Freezing cooler with metal block | Any | Store at -80°C | ||
Super-frost slides | Thermo Fisher Scientific | 9161155 | ||
Cover slips | Any | Recommend 24 X 40 mm | ||
Chloroform | Sigma-Aldrich | 288306 | Analytical grade | |
Glacial acetic acid | Merck | 100063 | Analytical grade | |
Ethanol | Merck | 100983 | Analytical grade | |
Methylbenzoate | Sigma-Aldrich | M29908-500G | Analytical grade | |
Paraplast plus | Sigma-Aldrich | 76258 | Paraffin | |
Tissue-Teck O.C.T. compound | Sakura | 4583 | ||
16% formaldehyde, methanol free | Polysciences | 18814 | ||
Glycerol | Sigma-Aldrich | G5150-1L |