Histological section refers to thin slices of tissue applied to a microscopic slide, usually around 5 to 10 micrometres thick, which are viewed under a microscope. For further discussion of histological section and staining methods, one should review histology article.
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The specimen is cut into the correct size and configuration prior to fixation and microtome cutting. The specimen is stained and positioned for proper orientation. With Mohs surgery or the CCPDMA method of cutting, the specimen is cut in a manner to allow mounting all of the surgical margins on one plane. With standard bread loafing, the specimen is usually cut into multiple sections with the surgical margin stained. Some technologist will stain the edge to be oriented toward the microtome. The cut specimen is then transferred directly to frozen medium for frozen section processing, or placed in small cassettes for dehydration and paraffin embedding.
Fixation is done either by the fixed tissue method with paraffin, or by frozen section. With fixed tissue method, the tissue specimen is preserve in either formaldehyde or an acidic solution until it is processed. The tissue is then removed from the preservative, dehydrated with multiple solvent baths, and fixed in hot liquid parafin. The hardened parafin block with the fixed tissue is then cut with the microtome. With frozen tissue sectioning, the tissue is immediately frozen prior to processing (frozen section).
The frozen tissue block embedded in a frozen cutting medium, or the paraffin fixed tissue is cut using a very fine knife called a microtome. A cryostat is a micotome mounted inside a freezer for processing frozen tissue.
The frozen thin slices of tissue are mounted on a warm glass slide at room temperature, or the paraffin embedded slides are mounted on a heated glass. This allow them to be stained and ready for staining. The tissue mounted slides are then dry in open air or in a drying oven.
Multiple stain baths are used to make the tissue more visible to the naked eye. Please see histology for discussion of the stains used. Sections usually have a very thin piece of glass applied over the surface called a cover slip. The glass cover slip is glued onto the slide with a special optical grade transparent glue.
| Stain | Common use | Nucleus | Cytoplasm | Red blood cell (RBC) | Collagen fibers | Specifically stains |
|---|---|---|---|---|---|---|
| Haematoxylin | General staining when paired with eosin (i.e. H&E) | Blue | N/A | N/A | N/A | Nucleic acids—blue
ER (endoplasmic reticulum)—blue |
| Eosin | General staining when paired with haematoxylin (i.e. H&E) | N/A | Pink | Orange/red | Pink | Elastic fibers—pink
Collagen fibers—pink Reticular fibers—pink |
| Toluidine blue | General staining | Blue | Blue | Blue | Blue | Mast cells granules—purple |
| Masson's trichrome stain | Connective tissue | Black | Red/pink | Red | Blue/green | Cartilage—blue/green
Muscle fibers—red |
| Mallory's trichrome stain | Connective tissue | Red | Pale red | Orange | Deep blue | Keratin—orange
Cartilage—blue Bone matrix—deep blue Muscle fibers—red |
| Weigert's elastic stain | Elastic fibers | Blue/black | N/A | N/A | N/A | Elastic fibers—blue/black |
| Heidenhain's AZAN trichrome stain | Distinguishing cells from extracellular components | Red/purple | Pink | Red | Blue | Muscle fibers—red
Cartilage—blue Bone matrix—blue |
| Silver stain | Reticular fibers, nerve fibers, fungi | N/A | N/A | N/A | Reticular fibers—brown/black
Nerve fibers—brown/black |
|
| Wright's stain | Blood cells | Bluish/purple | Bluish/gray | Red/pink | N/A | Neutrophil granules—purple/pink
Eosinophil granules—bright red/orange Basophil granules—deep purple/violet Platelet granules—red/purple |
| Orcein stain | Elastic fibres | Deep blue [or crazy red] | N/A | Bright red | Pink | Elastic fibres—dark brown
Mast cells granules—purple Smooth muscle—light blue |
| Periodic acid-Schiff stain (PAS) | Basement membrane, localizing carbohydrates | Blue | N/A | N/A | Pink | Glycogen and other carbohydrates—magenta |
Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstown, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3.
The Nissl method and Golgi's method are useful in identifying neurons.