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Preparation of the planarian Schmidtea mediterranea for high-resolution histology and transmission electron microscopy

Abstract

The flatworm Schmidtea mediterranea is an emerging model species in fields such as stem cell biology, regeneration and evolutionary biology. Excellent molecular tools have been developed for S. mediterranea, but ultrastructural techniques have received far less attention. Processing specimens for histology and transmission electron microscopy (TEM) is notoriously idiosyncratic for particular species or specimen types. Unfortunately, however, most methods for S. mediterranea described in the literature lack numerous essential details, and those few that do provide them rely on specialized equipment that may not be readily available. Here we present an optimized protocol for ultrastructural preparation of S. mediterranea. The protocol can be completed in 6 d, much of which is 'hands-off' time. To aid with troubleshooting, we also illustrate the major effects of seemingly minor variations in fixative, buffer concentration and dehydration steps. This procedure will be useful for all planarian researchers, particularly those with relatively little experience in tissue processing.

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Figure 1: Good fixation requires fresh glutaraldehyde and formaldehyde.
Figure 2: Mildly hypertonic buffers cause pronounced osmotic shrinkage during fixation.
Figure 3: Artifacts not caused by fixation.
Figure 4: Preservation of internal structures.
Figure 5: The dorsal body wall.

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Acknowledgements

We thank L.-A. Miller (Materials Research Laboratory) and S. Robinson (Imaging Technology Group, Beckman Institute) at the University of Illinois, and A. Dufresne (Department of Biological Sciences) at the University of Manitoba, for maintenance of the transmission electron microscope facilities used by the authors. This work was supported by Canadian Mennonite University (Dean's Research Fund and Faculty Research Grant to J.L.B.) and the US National Institutes of Health (R01 HD043403 to P.A.N.). P.A.N. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

All authors helped to conceive the project. A.P.V. optimized the protocol and prepared most of the specimen blocks from which the images in this paper were obtained. J.L.B. sectioned and imaged the specimens, and drafted the manuscript. All authors helped to revise the manuscript and approved the final version.

Corresponding author

Correspondence to John L Brubacher.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Specimens fixed in aged glutaraldehyde.

a-c: Light micrographs of thick (0.5 μm) longitudinal sections, stained with toluidine blue. The dorsal side of the worms is up. Unless otherwise noted, specimens were processed according to the standard protocol described in the paper, but using aged glutaraldehyde (stored 4 years at 4°C). Of 8 specimens examined, poor fixation was pronounced in the 3 thickest (>400 μm thick along the dorsoventral axis) of which a and b are examples, and only apparent in 1 of 5 specimens less than 400 μm thick. This latter specimen (350 μm thick) is shown in c. a,b: Arrowheads indicate poorly fixed regions, evident as weak staining due to loss of tissue components. The boxed region in a is illustrated in d at higher magnification. c: Boxed regions – well fixed on left, poorly fixed on right – are shown at higher magnification in f and g, respectively; lu: intestinal lumen – likely a channel for fixative penetration, which explains the better fixation in its vicinity. d: Higher magnification view of boxed region from a. The boxed region corresponds to that illustrated by the transmission electron micrograph in e. Asterisks indicate lipid droplets in intestinal phagocytes, whose distinctive greenish colour is due to osmication. This greenish colour is evident in residual material left in extracted lipid droplets (o, arrows) suggesting that osmium (secondary fixative) did penetrate to central areas of the worm, and poor fixation is therefore not due to inadequate osmication. e: Transmission electron micrograph of lead-stained, thin (70 nm) section, roughly 1 μm deep to boxed region in d. Electron-dense material is evident in the largely extracted lipid droplets (arrows, top-left inset), correlating with the osmiophilic residue indicated in d. Extraction of cytoplasmic ground substance is evident in the bottom inset, showing a muscle fibre (m) and cytoplasm of an adjacent intestinal phagocyte (cy), providing further evidence that the poor fixation is due to inadequate aldehyde fixation, rather than incomplete osmication. f,g: Higher magnification views of boxed regions in c (intestinal phagocytes). Material in f is well fixed, while much material has been lost from the region illustrated in g. Note the relative lack of basophilic material in the cytoplasm, and the weak staining of material in the cores of the storage granules (+) in panel g, as compared to f. The extraction of material from the cores of lipid droplets (*) in f is due to the dehydration procedure used for this specimen (see Figure 3) and not fixation per se. Scale bars: a-c: 200 μm, d, f, g: 20 μm, e: 6 μm (2 μm for insets).

Supplementary Figure 2 Illustrations for steps 5 and 18.

a: To minimize physical damage to worms in step 5, they should be cut after a brief initial fixation using a rocking “guillotine” motion using a scalpel with a rounded end. The rocking motion initiates cutting of tissue at the thinnest point of the worm's body, and reduces compression by minimizing the diameter of the worm in contact with the blade at any time. b: Drawing of one end of a wooden applicator stick. To produce a mini-spatula as described in step 18, carve one end of the stick into a thin, flat surface by whittling as indicated by the dashed lines in this image.

Supplementary information

Supplementary Figure 1

Specimens fixed in aged glutaraldehyde. (PDF 26005 kb)

Supplementary Figure 2

Illustrations for steps 5 and 18. (PDF 940 kb)

Supplementary Table 1

Chemical fixation methods described for resin embedding of Schmidtea mediterranea, 2010–present. (PDF 97 kb)

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Brubacher, J., Vieira, A. & Newmark, P. Preparation of the planarian Schmidtea mediterranea for high-resolution histology and transmission electron microscopy. Nat Protoc 9, 661–673 (2014). https://doi.org/10.1038/nprot.2014.041

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