2.3. Clearing

Clearing is the process of replacing the dehydrating fluid with another fluid that is totally miscible with both the dehydrating fluid and the embedding medium. The choice of a clearing agent depends upon the following:

i. The type of tissues to be processed and the type of processing to be undertaken

ii. Intended processing conditions, such as temperature, vacuum and pressure

iii. Safety factors

iv. Cost and convenience

v. Speedy removal of dehydrating agent

vi. Ease of removal by molten paraffin wax

vii. Minimal tissue damage

Commonly used clearing agents include toluene, xylene, chloroform, benzene, etc.

 

2.4. Embedding and Sectioning

After tissues have been dehydrated and before they can be "sectioned" i.e. sliced very thinly, they must be secured in a very hard solid block in such a way that the hardened material secure all parts of the biological tissues and is transparent to the optical method used for viewing the finished samples. Generally, wax, polyethylene glycol (PEG), or resins (e.g. LR white) are used as embedding material for histology. During this process the tissue samples are placed into moulds along with liquid embedding material which is then hardened.

Paraffin wax is probably the most commonly used embedding material, being easy to section. It is a polycrystalline mixture containing solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds of the density and slightly more elastic than dried protein. Wax and polyethylene glycol (PEG) are removable matrix; PEG is water soluble and requires lower infiltrating temperature than wax. Resins are not easily removable, however, they produce a much harder block and, therefore, thinner sections can be obtained.

Sectioning an embedded tissue sample is the next step necessary to produce sufficiently thin slices of sample that the detail of the microstructure of the cells/tissue can be clearly observed using microscopy techniques (either light microscopy or electron microscopy). Sectioning of embedded tissues depends on the type of microscopy that will be used to observe it and, hence, the thickness of sample required. In the case of samples to be studied using light microscopy, a steel knife mounted in a microtome may be used to cut 10μm thin sections, which are then mounted on a glass microscope slide. In case of samples to be studied using transmission electron microscopy, a diamond knife mounted in an ultramicrotome may be used to cut 50 nm thin sections, which are then mounted on a 3-millimeter-diameter copper grid. A microtome is a mechanical device utilized to slice biological specimens into very thin segments for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of plant tissues for histology.