2.2 Embedding and sectioning
Tissue that has been received in the laboratory needs to be prepared for sectioning. A variety of instruments are used to cut the sections and the protocol depends on the application. In most cases the tissue requires embedding in a medium, which allows thin sections to be cut cleanly; most tissues for routine histology are embedded in wax blocks. This requires that water is removed from the tissue and progressively replaced by wax, which can be solidified later to make a tissue block suitable for sectioning. The tissue is progressively dehydrated by immersing it in successively higher concentrations of alcohol (ethanol), before transfer to the organic solvent xylene and finally embedding in wax. Xylene is used at the final stage because wax is soluble in xylene, but not alcohol, so the wax can readily permeate the tissue. In a large pathology laboratory, much of this tissue processing is automated in order to save time and to produce consistent results.
A number of devices are available for cutting sections:
- Microtome cuts thin sections (1-50µm) from fixed, embedded tissue (1µm =10-6 metres)
- Vibratome uses a vibrating blade to cut thicker sections from fresh or fixed tissue (up to 200µm).
- Cryostat cuts sections from deep-frozen blocks of unfixed tissue.
Most sectioning in routine histopathology departments is done with a microtome producing sections of ~3µm thickness, from tissue that has been embedded in wax.
How thick is a typical cell in relation to the thickness of a 3µm section? How many cells might be seen by focusing up and down through the full depth of a section?
Most cells are >3µm in thickness, although it depends on the cell type and its shape. Hence a section will be less than one cell thick.
The vibratome and cryostat are often used to cut unfixed sections, and these are often more suitable for antibody staining, but they are not the first choice for routine sectioning (antibodies are expensive and the staining procedure takes several hours. H&E is inexpensive and the staining can be done within minutes). Normally a H&E-stained section is examined first, before deciding whether additional tests or staining procedures are required. Even then, the antibody-staining is normally done on wax-embedded sections, using antibodies that are known to work on fixed sections.