How to Prepare Histology Slides

1. Fixing

Samples of biological tissue are 'fixed' to preserve the cells/tissue in as natural a state as possible and prevent postmortem decay (autolysis and putrefaction). Chemical fixatives are very carefully selected substances whose properties must meet many criteria. Even the most careful fixation alters the sample to a certain extent and may potentially introduce artifacts that can interfere with interpretation of images of the fine detail of cells, incl. all their organelles, that can only be observed using an electron microscope (such fine detail of cells that can only be seen using electron microscopes is called 'cellular ultrastructure').
Part of the skill involved in preparing and interpreting histology slides is in maintaining the structure of the sample in as undisturbed a state and possible, minimising the introduction of artifacts and recognising artifacts as such when they are present. Because fixation is usually the first stage in a multistep process to prepare biological material for microscopy or other analysis, the choice of fixation method and specific fixative may depend on the subsequent processing steps appropriate in that particular case.

Chemical Fixation.
In this case biological structures are preserved (both chemically and structurally) in a state as close to that of the living tissue as possible. This requires a chemical fixative that can stabilise the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble.

Frozen Sections
Small pieces of tissue (typically 5mm x 5mm x 3 mm) are placed in a cryoprotective embedding medium then snap frozen in isopentane (an alkane) cooled by liquid nitrogen. Tissue is then sectioned in a freezing microtome or cryostat. Sections are then fixed by immersion in a specific fixative or series of fixatives for carefully controlled period of time.

2. Processing

Tissue processing is done to remove water from the biological tissues, replacing such water with a medium that solidifies, setting very hard and so allowing extremely thin sections to be sliced. This is important because biological tissue must be supported in an extremely hard solid matrix to enable sufficiently thin sections to be cut. Some typical values are:

Removal of water is 'dehydration'.

3. Embedding

After tissues have been dehydrated and before they can be "sectioned" i.e. sliced very thinly (see the thicknesses mentioned above) they must be secured in a very hard solid block in such a way that the hardened material used to secure all parts of the biological tissues in place is transparent to the optical method used for viewing the finished samples.

Different types of embedding techniques and materials are used depending on the sample being prepared and the other types of processing involved in preparing that particular sample.

In general, tissue samples are placed in molds together with liquid embedding material which is then hardened. The result of this stage in the preparation of histology slides is hardened blocks containing the original biological samples together with other substances used so far in the preparation process.

4. Sectioning

Sectioning an embedded tissue sample is the 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).

Possible orientations at which tissue samples may be sectioned include:

• Vertical sectioning perpendicular (i.e. at right-angles) to the surface of the tissue.
  This is the most common method.
• Horizontal sectioning is often done for the study of hair follicles and structures that include hairs, hair follicles, arrector pili muscles, and sebaceous glands in general. Such structures are sometimes called "pilosebaceous units".
• Tangential to horizontal sectioning is done in chemosurgery (also called 'Mohs surgery') which is a form of microscopically controlled surgery used to treat certain types of skin cancer.

The method used to actually cut sections from the hardened block of tissue 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 tissue sections which are then mounted on a glass microscope slide. In the case of samples to be studied using transmission electron microscopy, a diamond knife mounted in an ultramicrotome may be used to cut 50 nm tissue sections which are then mounted on a 3-millimeter-diameter copper grid.

5. Staining

Finally, the mounted sections are treated with an appropriate histology stain.

Why are histology samples stained ?

Put another way, what is the purpose of histology stains ?

Biological tissue has very little variation in colours/shades when viewed using either an ordinary light (optical) microscope or an electron microscope. Staining biological tissues is done to both increase the contrast of the tissue and also highlight some specific features of interest, depending on the type of tissue and stain used.