Sample Preparation
In the fixation stage, a fixative is used to modify tissue by stabilizing the protein so it is resistant to further changes; thus, fixatives are used to protect tissue from degradation and to maintain the structure of the cells, including sub-cellular components such as cell organelles (nucleus, endoplasmic reticulum, and mitochondria). In the processing stage, tissues must go through a dehydration process to replace water with the material that will be used for embedding, so the tissue samples adhere to the embedding material.
The most common technique is paraffin wax embedding. Samples are placed in tissue cassettes and then immersed in multiple baths of progressively more concentrated alcohol to remove water from the tissues. This is followed by a clearing agent, such as xylene, to remove the alcohol and finally hot molten paraffin wax, which replaces the xylene and infiltrates the tissue.
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Embedding and Sectioning
In the embedding process, tissue samples in their cassettes and liquid embedding material (such as paraffin wax) are placed into molds and allowed to harden. The hardened blocks containing the tissue samples are then ready to be sectioned. Embedding can also be accomplished using frozen, non-fixed tissue in a water-based medium, usually a water-based glycol or resin.
In the sectioning phase, the tissue is sliced into very thin (3-5µm; 1000µm = 1mm) sections using a microtome. These slices, usually thinner than the average cell, are then placed on a glass slide for staining. Frozen tissue embedded in a freezing medium is cut on a microtome in a cryostat.
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Staining and Imaging
Routine staining is done to give contrast to the tissue being examined. Haematoxylin and Eosin (H and E) are the most commonly used stains in histology.
Haematoxylin is combined with a metal mordant, such as aluminum, and the haematoxylin-aluminium complex is used to stain cell nuclei blue/black. Eosin is the most widely used counterstain in the routine staining of tissue sections. The best staining with eosin occurs at pH 4.5 to 5 and, when used properly, multiple shades of pink can be obtained. Erythrocytes, collagen, and the cytoplasm of epithelial or muscle cells should stain different shades of pink when using eosin. After staining the sections need to be mounted to protect the specimen, which requires the use of mounting media and a coverslip.
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Storage
In histology there is a requirement to store the paraffin tissue block and the stained microscope slide. The Fisher Scientific channel has a range of storage devices to offer.
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