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Making histological sections for the light microscope

The paraffin technique is most commonly used to prepare tissue sections for microscopic examination. In this technique, tissues are fixed, processed and embedded in wax. Sections are made from the wax blocks and then stained to help distinguish the components of the tissue using light microscopy. The optical densities of the different tissue elements in unstained tissue sections are very similar, so dyes are used to impart colour to the tissues to aid in their visualisation.

1. Fixation

Tissue samples are usually fixed by chemical fixation, using formaldehyde-containing fixatives which bind to and form cross-links with proteins and DNA, while alcoholic fixatives denature and precipitate proteins through dehydration. Fixation hardens the tissue, and inactivates enzymes that might otherwise degrade the tissue. Fixation also kills bacteria, thus preventing bacterial decomposition and putrefaction. Some fixatives can also enhance tissue staining. The fixative most commonly used is 10% neutral buffered formalin.

2. Dehydration and clearing

By gradually replacing water in the tissue sample with alcohol, dehydration of the tissue is achieved by passing the tissue through increasing concentrations of ethyl alcohol (from 0 to 100%). In order to cut sections, tissue blocks must be embedded in paraffin wax, which acts as a support medium. Because wax is immiscible with water and alcohol, a suitable intermediate reagent is required. The paraffin solvent called 'xylene' is used to remove the alcohol from the tissue. The alcohol is then replaced with xylene, which is miscible with both alcohol and wax. This final step to remove the alcohol from the tissue is called 'clearing'.

3. Embedding

The tissue is placed in warm paraffin wax, that infiltrates into and around the tissue. Samples are then orientated in a metal mold which is filled with molten wax. After cooling the tissue block to harden it, thin slices (sections) of tissue can be cut on a cutting instrument called a microtome.

4. Sectioning

The tissue is trimmed, and mounted on a rotary microtome (shown in the picture). Thin sections are cut, stained with a routine stain and mounted on a microscope slide. The slide is now ready for viewing under the light microscope.



Are there any other ways of making sections for light microscopy?

There are two further techniques that are sometimes used:

  1. Frozen sections - Tissues are frozen rapidly in liquid nitrogen, or dry ice and then cut in a refrigerated cabinet (cryostat) with a cold knife, stained and observed under the microscope. This procedure is faster, and can preserve some tissue details that may be lost by the paraffin technique. Sections are  cut at 5 - 10 µm thickness.
  2. Semithin sections - It is sometimes hard to see detail in thick sections. To get around this, sections can be embedded in epoxy or acrylic resin, which enable thinner sections (less than 2 µm) to be cut.