Pre-Embedding Antibody Staining for TEM
by Hall, Hartwieg and Nguyen


Summary Protocol
  1. Collect cryo-thin sections onto 2.3 M sucrose drops held in a wire loop. They will freeze seconds after pickup while in the cryomicrotome chamber.
  2. Remove wire loop from the chamber and allow the sections to thaw. Then move these sections from sucrose droplets onto carbon-coated Pioloform (or Formvar) nickel grids (see Grid making). These grids can then be stored on 0.3% polylysine coated glass slides.
  3. Move the slides containing nickel grids into a wet chamber as soon as possible, so the sections never dry.
  4. Antibody staining should be done in a humidified chamber, starting with a blocking step. Times and concentrations depend on the antibody and epitope (cf. van Donselaar et al., 2007; Ripper et al., 2008).
  5. After the antibody staining, grids are rinsed in buffer.
  6. Stain each grid in a solution of 1.8% methylcellulose + 0.3% uranyl acetate.

Description

The development of techniques for applying gold-linked markers and antibodies at the TEM level has occupied workers for many years. Important developments follow early work of Tokuyasu (Martins-Green and Tokuyasu, 1988; Tokuyasu, 1973, 1986) and Slot and Griffiths (Liou et al., 1996) for pre-embedding techniques.

Early work on this method for nematode tissues was conducted by Selkirk et al. (1991) and more recently by Willisa Liou and colleagues (cf. Liou et al., 1996; Sato et al., 2005, 2008). The protocol shown here reflects the general method followed by Hartwieg and by Liou and colleagues (Summary protocol). A detailed protocol for antibody treatments of cryo-thin sections on nickel grids has been published by Slot and Geuze (2007).

Helpful Hints

Primary antibody can be followed by a gold-conjugated Protein A (Sato et al., 2005; Slot and Geuze, 2007), or a gold-linked secondary antibody. It is also feasible to use an ultra-small Nanogold-linked secondary antibody, followed by silver enhancement (Ripper et al., 2008).

Troubleshooting

Compare several different dilutions of the primary antibody (see Hall, 1995; Paupard et al., 2001) to optimize specific binding versus nonspecific background binding. The primary antibody used in these experiments should be immunopurified prior to use to reduce nonspecific binding.


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

Liou, W., Geuze, H.J. and Slot, J.W. 1996. Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106: 4158. Article

Martins-Green, M.M. and Tokuyasu, K.T. 1988. A pre-embedding immunolabeling technique for basal lamina and extracellular matrix molecules. J. Histochem. Cytochem. 36: 453458. Article

Paupard, M.-C., Miller, A., Grant, B., Hirsh, D. and Hall, D.H. 2001. Immuno-EM localization of GFPtagged yolk proteins in C. elegans using microwave fixation. J. Histochem. Cytochem. 49: 949–956. Article

Ripper, D., Schwarz, H. and Srierhof, Y.-D. 2008. Cryo-section immunolabelling of difficult to preserve specmens: advantages of cryofixation, freeze-substitution and rehydration. Biol. Cell 100: 109123. 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

Tokuyasu, K.T. 1973. A technique for ultracyotomy of cell suspensions and tissues. J. Cell Biol. 57: 551565. Article

Tokuyasu, K.T. 1986. Application of cryoultramicrotomy to immunocytochemistry. J. Microsc. 143: 139149. Abstract

van Donselaar, E., Posthuma, G., Zeuschner, D., Humbel, B.M. and Slot, J.W. 2007. Immunogold labeling of cryosections from high-pressure frozen cells. Traffic 8: 471485. Article


image