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Appendix
useful for endocrine organs in which cells that produce different secretions can be stained different
colors.
Wright stain
is also a mixture of stains containing one acidic dye (methylene blue) and one basic
dye (eosin). Wright stain is widely used for blood and bone marrow smears. Basophilic granules are
deep purple, acidophilic (or eosinophilic) granules are reddish, and the cytoplasm is usually bluish gray,
although it may appear pink to red (or mottled) if it contains acidophilic substances.
Elastic f
ber stains
are individual or combinations of dyes that stain cellular constituents but will
also prominently stain elastic F
bers. These exploit relatively nonspeciF
c ionic and nonionic differences
between collagen and elastin. Depending on the procedure used, elastic F
bers may appear brown, deep
purple, blue-black, or black.
Reticular f
ber stains
, as is the case for elastic stains, also exploit the relatively nonspeciF
c differ-
ences in the amount of glycosylation of reticulin versus that of collagen. In general, reticular F
bers are
stained black, using silver as the chromophore, with the other cellular elements appearing as a mono-
chromatic background of gray or various shades of light red. Whereas reticular F
bers stand out in these
special stains, other cellular detail does not.
Silver stains
represent a large category of what are actually silver impregnation methods, not
stains. In general, these methods use dilute solutions of silver nitrate (gold and mercury are alse some-
times used) to impregnate blocks of nerve tissue and precipitate the metal ions on the cell membranes
of neurons and glia. Tissue blocks, generally no more than 0.3 to 0.6 cm thick, are F
xed in formalin,
treated with a mordant solution such as potassium dichromate, suspended in a dilute silver nitrate
solution for several days (with block cleaning and solution changing every day), and then encased in
parafF n (not embedded). Sections are cut at thicknesses ranging from 40 to 70
m
m and mounted on
slides. Individual neurons and glia cells appear black on a light-yellow to off-white background. The
mechanism of attachment of the silver ions to the cell membranes is not well understood.
The
periodic acid-SchiFF (PAS)
reaction is a method that is more speciF
c in its staining reac-
tion than the anionic, cationic, or lipid soluble dyes. One such stain is the Schiff reagent (leucoba-
sic fuchsin), which reacts speciF cally with free aldehydes. Pretreatment with periodic acid converts
adjacent hydroxyl groups, such as those found in glycogen, into aldehydes. Treatment with the Schiff
reagent stains the free aldehyde groups red; the sections are usually counterstained with H&E. The PAS
reaction demonstrates sites of high concentrations of polysaccharide-containing components, such as
glycogen and glycosaminoglycans.
Immunocytochemistry
is a specialized method that can be used to precisely localize enzymes or
large molecules (macromolecules) within the cell or on its membrane. The immune system of the body
is able to defend itself against foreign molecules (antigens) by producing speciF
c types of proteins (anti-
bodies). Immunocytochemistry methods use this feature of cells to visualize speciF
c molecules. ±or light
microscopy, an antibody is produced (for example, in rabbit tissue) against a speciF
ed protein or mol-
ecule, and the antibody is coupled with a dye, such as fl uorescein or rhodamine B. When this labeled
antibody attaches to a speciF c antigen and is exposed to ultraviolet light, it will fl
uoresce greenish yel-
low (fl uorescein) or bright red (rhodamine B), thereby speciF
cally identifying the location of that mol-
ecule. This is the direct method: An antibody is produced, coupled to a dye, attached to an antigen, and,
thus, becomes visible. In the indirect method, unlabeled antibodies are produced in one animal (rabbit)
against a speciF c antigen and then applied to a tissue to which they attach. The unlabeled antibodies are
visualized by exposing them to labeled antibodies that are made in another species (goat) and that are
directed against the immunoglobulins from the F
rst species. One way to visualize this is as follows: An
antibody is produced, not labeled, and attached to an antigen; a second antibody is produced, coupled
with a dye, attached to the F rst unlabeled antibody, and, thus, then becomes visible.
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