Preparation of Tissues and Cells for Immunocytochemistry
Successful immunocytochemistry is only possible when antibodies (or other affinity markers) are able to bind to their target ligands. For extracellular antigens this is not a problem, but for molecules inside cells the affinity markers must gain access. The best way to gain access into cells, for electron microscopy, is to cut sections through them. To prepare thin sections through biological material is not a simple process. Usually the water within the material is replaced with a medium that can be hardened, either by freezing or by polymerization.
The cryosection technique (Tokuyasu 1973, 1976, 1986; Tokuyasu and Singer 1976; Griffiths 1984, 1993; Griffiths et al 1983) is now one of the two most important techniques for subcellular immunocytochemistry, the other being resin embedment. Cryosectioning has been applied to the localization of many different antigens and for some antigens it appears to be the most sensitive detection method available (Griffiths and Hoppeler, 1986). An extra advantage is that semi-thin sections cut from the same block as that used for thin sections can be labeled with fluorescent or gold markers for a light microscopic overview of the labeling pattern.
- Tokuysau, K. T. 1973.
- A technique for ultracryotomy of cell suspensions and tissues. J. Cell. Biol. 57:551-565.
- Tokuysau, K. T. 1976.
- Membranes as observed in frozen sections. J. Ultrastruct. Res. 55:281-287.
- Tokuysau, K. T. 1986.
- Application of cryoultramicrotomy to immunocytochemistry. J. Microsc. 143:139-149.
- Tokuyasu, K. T. and J. S. Singer. 1976.
- Improved procedures for immunoferritin labelling of ultrathin frozen sections. J. Cell Biol. 71:894-906.
- Griffiths, G. and H. Hoppeler, 1986.
- Quantitation in immuncytochemistry, correlation of immunogold labelling to absolute numbers of membrane antigens. J. Histochem. Cytochem. 34:1389-1398.
- Griffiths, K. Simons, G. Warren and K. T. Tokuyasu. 1983.
- Immunoelectron microscopy using thin frozen sections: applications to studies of the intracellular transport of Semliki Forest virus spike proteins. Methods Enzymol.96:466-484.
- Griffiths G. 1984.
- Selective contrast for electron microscopy using thawed frozen sections and immunocytochemistry. In: Revel, J. P., T. Barnard, G. H. Harris (eds). Proc. Congr. Specimen Preparation. Traverse City, Michigan, SEM Inc., Chicago, pp153-159.
- Griffiths, G. 1993.
- Fine structure immunocytochemistry. Springer Verlag, Heidelberg.
Practical Details for Cryosectioning.
The procedure for cryosectioning is relatively simple if a few technical rules are observed and can be performed, from start to finish, in one day. Before starting, however, some specialized equipment and chemicals are required. Once these have been obtained it should now be possible to follow the following procedural outline;
Biological material is fixed, usually by immersion in a buffered aldehyde solution. The material is then cut into small pieces, infused with a sucrose solution, which acts as a cryoprotectant, and then placed onto small specimen pins. The pins, with a specimen on, are frozen by immersion in liquid nitrogen, quickly transferred to a cooled cryochamber fitted on an ultramicrotome and sectioned. The sections are obtained using a dry glass or diamond knife and collected on the knife surface. The sections are retrieved from the knife by picking them up on a small drop of a suitable solution (usually sucrose) and transferred onto formvar/carbon-coated specimen grids. The grids are floated on buffered saline with the section-side in the liquid. The back of the grid is kept dry at all times. The sections on the grids are immunolabeled and incubated with a gold visualization probe. Finally the sections are dried in the presence of a thin plastic film to avoid surface tension damage.
- Cryoprotection and Freezing.
- Section retrieval.
- Contrasting and Drying.
- EM Examination and Evaluation.