|Atlas||Wheater’s Cell Structure and Function (review)|
|Text||Ross & Pawlina, Chapters 1-3, pgs 1-91 (review)|
From the lecture
From the lab session
Hematoxylin and Eosin
Hematoxylin is the most commonly used nuclear stain in histology and pathology although, despite its long use and honorable history, the chemistry of the dye is still not fully understood. Essentially, hematoxylin is a basic dye and complexes with nucleic acids (DNA and RNA in the nucleus; RNA in the cytoplasm) or other negatively charged molecules (such as sulfate groups). Structures that bind hematoxylin are therefore termed “basophilic” (base loving).
Eosin is an acidic dye and the basic structures it stains are termed “eosinophilic” or less commonly “acidophilic” (acid loving). It stains membranes and most proteins. Cells that have large quantities of folded membranes stain intensely with eosin, because of basic amino acids in the membranes (e.g. macrophages contain lots of membrane in the form of phagocytic vesicles as well as basic lysosomal enzymes within those vesicles that stain with eosin). Collagen is generally stained some shade of red/orange whereas actin (such as in smooth muscle cells) is a bit more pink. Elastin, when present in relatively large amounts (such in the walls of blood vessels, in elastic cartilage, and in the esophagus and trachea), will appear glassy red.
A note about acids/bases and their charges: It always seems to a point of confusion as to how it is that an acid such as DNA can have a negative charge when we generally think of something that is acidic as being positively charged (i.e. a solution with lots of H+ ions is “acidic”). However, the better way to think of acids is as proton donors –in solution, an acid such as DNA donates H+ protons (which makes the solution acidic). Upon donating protons, the DNA therefore becomes negatively charged and it is in this state that it binds hematoxylin.
Masson Triple Stain (or “Trichrome”)
This dye combination stains mucus as well as collagenous and reticular fibers blue (aniline blue) or green (fast green) depending on the mixes of dyes used; muscle red; nuclei red (they are black if preceded by an iron hematoxylin). This is a commonly used connective tissue stain in both histology and pathology. On your slides the stain is designated “Masson” or “Mass”; but the blue or green collagen is the tip-off.
In this case silver nitrate is reduced to metallic (black) silver. The process of development and fixation is similar to developing a photograph (stains reticular fibers).
Periodic Acid Schiff (PAS)
This is an extremely useful technique for demonstrating glycoproteins, mucins and some proteoglycans -anything that contains a relatively high amount of sugar groups. It involves the generation of dialdehydes from hexoses (present as the carbohydrate portion of the aforementioned compounds. One of its main uses is the demonstration of basement membranes, especially in the kidney, and/or in sections with epithelia atypia, where breech of the basement membrane is suspected in early carcinomas. An excellent example is slide 210 from the kidney WebScope ImageScope where PAS staining demonstrates the basement membranes (pink lines) of the simple cuboidal epithelium lining the tubules and squamous epithelium in the glomeruli (the round tangles of cells). Note that PAS staining also shows the glycocalyx associated with microvilli (appears as a fuzzy pink border) on epithelia lining some of the tubules.
Click the”Virtual Micrscopy Link” in the menu bar to find more information about virtual slides: Set up to access the virtual slides on campus; Installing Aperio ImageScope; Navigating digital slides using Aperio Imagescope; Using Aperio Imagescope Annotations; Using WebScope to Create “Bookmarks” to Regions of Interest
One of the main advantages of viewing the virtual slides in ImageScope is the ability to annotate the images in a “layer” that you can then save to your own computer (the original image on the server is not altered in any way).