Method for the Preparation of a Histology Section

The mainstay of immunohistochemistry and pathological diagnosis is the use of the traditional light microscopy. In order to view tissue and individual cells, sections from tissue must be thin enough to allow light to pass through them and flow the image to the other optic. Unfortunately, the majority of tissues are far too soft and fragile to be cut thinly and therefore are immersed and absorbed with a more rigid and hardening solution which may support the matrix of the tissue, such as wax (paraffin) or plastic. These components are both repelled by water, and therefore water must be removed from tissue prior to supporting the matrix. Removal of water occures by gradual substitution with 100% alcohol. The alcohol is subsequently substituted also by toluene or xylol. These agents are soluble with both paraffin wax and plastic. The paraffin technique is the most widely used method of preparing tissue for histological examination with light microscopy.

The Paraffin Technique

The paraffin technique involves 5 steps: fixation, dehydration, clearing, infiltrating and embedding and sectioning.

Fixation

Fixation of tissue samples with formalin occurs almost immediately in order to prevent any deterioration and/or degradation of cells.

Dehydration

As mentioned previously, tissue samples are gradually passed through 100% alcohol in order to remove and replace all the water with alcohol.

Clearing

This step is required to replace the alcohol with xylol.

Infiltrating and Embedding

Paraffin wax is then used to replace the xylol.

Sectioning

This is the last step. The treated tissue sample is finally sections/cut with a microtome into thin, transparent sections that are between 5 to 10 micrometres thin.