Notre Dame Integrated Imaging Facility

Sample Preparation and Staining Information References

Sample Preparation and Staining Information References

Histology sample preparation prepares tissue specimens for sectioning, staining and diagnosis. The standard paraffin process (tissue processing) moves specimens through a series of steps so the soft tissue is supported in a medium that allows sectioning. The standard steps are: Fixation that preserves the tissue, Processing that dehydrates, clears and infiltrates the tissue with paraffin wax, Embedding that allows orientation of the specimen in a “block” that can be sectioned and is easy to store and handle, and Sectioning using a microtome to produce very thin sections that are placed on a microscope slide ready for staining. Frozen sectioning is an alternative preparation technique that quickly freezes tissue to preserve it and provide sufficient hardness so it can be sectioned immediately using a cryostat. This technique is often used during surgery where the surgeon needs to locate a tumor margin to ensure it has all been removed.

Routine &Special Staining

Routine (H&E) staining is the corner stone of tissue-based diagnosis. The process stains thin tissue sections so that pathologists can visualize tissue morphology. The process uses a haematoxylin dye to stain cell nuclei (and other parts) blue and an eosin dye to stain other structures pink or red. Properly applied, this technique provides exceptional detail of tissue structure and the makeup of the cells. This detail is required for tissue-based diagnosis, particularly in the detection and classification of cancer. Special stains use a variety of dyes and techniques to stain particular tissues, structures or pathogens (such as bacteria) to assist pathologists with tissue-based diagnosis.

IHC & ISH Advanced Staining

When morphology and routine staining cannot provide all the diagnostic answers, pathologists turn to advanced staining techniques: Immunohistochemistry (IHC) and in situ hybridization (ISH). IHC detects the presence of critical marker proteins in tissue samples while ISH detects target RNA or DNA sequences. Both techniques are commonly used for cancer diagnosis where the presence or absence of particular proteins or sequences help pathologists make an accurate diagnosis and differentiate between disease states that look morphologically similar. IHC uses primary antibodies that bind to the target protein and detection systems that link to the primary antibody to provide a visual indication of the protein’s presence and location using brightfield microscopy. ISH uses probes that bind to the target RNA or DNA sequence. Different detection systems are then used to visualize the presence of the target sequence: FISH uses fluorescent dyes and fluorescent microscopy while CISH uses chromogenic dyes and brightfield microscopy.