Cryo-Thin Sectioning
by Hall, Hartwieg and Nguyen


Summary Protocol
  1. Prepare a primary fixation solution as follows: 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2.
  2. Wash live worms off the culture plate into a 9 well Pyrex dish with the fixative.
  3. Cut worms open with a razor blade while in fix.
  4. Move the worms in fixative to the refrigerator to continue fixation overnight.
  5. Embed worms in 12% gelatin (or agar), grouping them together. Allow gelatin to cure in the refrigerator for several hours.
  6. Transfer into 15% PVP + 1.7 M sucrose; place samples on a shaker for up to 24 h in the cold room to allow complete exchange of solutions.
  7. Cut gelatin under the dissection microscope into small blocks as tall trapezoids with a wide base.
  8. Place each block onto a microtome pin for cryo-sectioning and freeze the blocks rapidly in liquid nitrogen.
  9. Store frozen blocks on microtome pins in holey Eppendorf tubes (to allow flow-through of nitrogen) under liquid nitrogen until ready to cut.

Preparations Prior to Collection of Cryo-Thin Sections

Currently, the use of frozen thin sections of the nematode is not very common within the C. elegans community, as it is technically difficult and requires more specialized equipment. However, it may prove to be very useful in combination with a modern TEM, using an environmental chamber and a cryo-stage for molecular studies in situ, either for something akin to crystallography of a molecule’s resting shape, or for an understanding of macromolecular assemblages. More often, this method has been used as part of a pre-embedding immunoEM approach (Sato et al., 2005, 2008; Selkirk et al., 1991).

There are several potential advantages to utilizing cryo-thin sections for immunoEM. Firstly, one avoids any use of dehydration or plastic resins, either of which may degrade the epitope under study. Secondly, the epitope itself may be somewhat better exposed to the primary antibody in a cryo section than when buried in a plastic thin section. One still must take into account the sensitivity of the epitope to the fixative. We have previously reviewed how to test this sensitivity prior to embarking on immunoEM protocols (Hall, 1995; Paupard et al., 2001).

Helpful Hints
Worms can be anaesthetized in M9 prior to fixation if desired. The best fixative for immunoEM will depend upon the epitope in question (Hall, 1995). Alternate fixatives used for cryo-thin sections have included 4% paraformaldehyde in phosphate buffer, or 2.8% glutaraldehyde + 2% acrolein in cacodylate buffer, or 2% glutaraldehyde in caco dylate (cf. Sato et al., 2005, 2008). Total fixation time for some epitopes might be much shorter than the example shown in Summary protocol. Two hours at room temperature or 1 h at 4°C have also proven sufficient in some cases (Sato et al., 2005, 2008). For the shortest protocols, it is advisable to place the samples on a shaker to improve penetration of the fixative. One simple way to group the worms is to wash them into an Eppendorf tube after step 4, rinse in buffer, decant the wash buffer after spinning lightly to pellet the worms, add 0.5 mL of warm agarose (or gelatin), vortex the tube to mix the agar, then spin weakly again to gently pellet the worms. Cool this sample in the refrigerator for 10 min. The tip containing the pellet can then be cut into small blocks containing many worm pieces.

Troubleshooting
The gelatin (or agar) pad must have a relatively high concentration (12%) in order to give it enough physical integrity to hold sections together during frozen sectioning. Otherwise the worm cross-sections tend to separate from the frozen sections as they emerge on the knife edge and the worm slices will disappear into space, leaving behind an empty agar section with holes where the worms once laid.

Cutting Cryo-Thin Sections

In preparation to collect frozen thin sections, one requires a specialized ultra microtome with a ‘‘freezing stage’’ (knife and block must both be kept at very low temperature). Thin sections may be collected either on freshly broken glass knives, or on a cryo-diamond (Diatome). The knife holder and knife are held at very low temperature (–110°C), and the sample on its frozen pin is held on the end of the microtome arm, using a separate temperature control to maintain the sample at –100°C during the cutting action. Frozen samples are cut ‘‘dry’’ onto the frozen knife edge, where they collect as glistening small objects within an enclosed (chilled) cutting chamber. The best sections usually show a gold color. Frozen thin sections are transferred individually onto a small-wire loop containing 2.3 M sucrose. As the loop is moved close to the knife edge, sections will spontaneously leap off the edge of the knife and into the loop of sucrose, without direct mechanical force to move them. Once collected in the loop, individual sections are quickly treated with antibody and/or stains as explained below in Pre-Embedding Antibody Staining for TEM. During or after these treatments, stained sections are finally transferred to Formvar-coated nickel grids (mesh grids or slot grids) for examination under the TEM.

Helpful Hints
An alternate transfer solution is 1% methylcellulose + 1.2 M sucrose (cf. Sato et al., 2005). Pick up sections with a drop of sucrose on the loop and place the drop with the floating sections on top of the coated grid, so that the sections directly touch the grid coating. Place each grid upside down onto an agarose pad in a large Petri dish, so that the sucrose can fuse into the agarose. This can also be done by placing the grids on a buffer solution, but using the agarose step is gentler. For a more detailed protocol for cryo-sectioning, see Slot and Geuze (2007).


References

Hall, D.H. 1995. Electron microscopy and three-dimensional image reconstruction. Methods Cell Biol. 48: 395-436. Abstract

Hall, D.H., Hartweig, E. and Nguyen, K.C.Q. 2012. Modern electron microscopy methods for C. elegans. Methods Cell Biol. 107: 93-149. Abstract

Paupard, M.-C., Miller, A., Grant, B., Hirsh, D. and Hall, D.H. 2001. Immuno-EM localization of GFP- tagged yolk proteins in C. elegans using microwave fixation. J. Histochem. Cytochem. 49: 949–95. Article

Sato, M., Sato, K., Fonarev, P., Huang, C.J., Liou, W. and Grant, B.D. 2005. C. elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit. Nat. Cell Biol. 7: 559–569. Abstract

Sato, M., Sato, K., Liou, W., Pant, S., Harada, A. and Grant, B.D. 2008. Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME4, a coated pit protein. EMBO J. 27: 1183–1196. Article

Selkirk, M.E., Yazdanbaksh, M., Freedman, D., Blaxter, M.L., Cookson, E., Jenkins, R.E. and Williams, S.A. 1991. A proline-rich structural protein of the surface sheath of larval Brugia filarial nematode parasites. J. Biol. Chem. 266: 11002–11008. Article

Slot, J.W. and Geuze, H.J. 2007. Cryosectioning and immunolabelling. Nat. Protocols 2: 2480–2491. Abstract


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