Appendix
427
bubbles are removed by vacuum. Second, the tissue sample is oriented in an embedding mold. The mold
is then f lled with melted medium and allowed to cool so the medium hardens. A similar set oF steps are
Followed when a polymer is used as the embedding media For electron microscopy.
A comment on frozen sections:
As noted above, rapid Freezing can be used to f
x tissue. ±reezing
can also be used to prepare tissues For sectioning. Small pieces can be rapidly Frozen, mounted on a
microtome, and sectioned. Larger pieces can be immersed in a sucrose solution until Fully impregnated
with the sucrose (the tissue initially fl
oats and then sinks). The sucrose expedites the Freezing process
and renders minute ice crystals. The tissue is then mounted, Frozen, and sectioned.
Sectioning and Mounting
Sectioning oF prepared tissues is done on microtomes that are designed to accommodate sections
mounted in paraplast (paraFf
n), Frozen in ice, or embedded in plastic. The sections are cut using
extremely sharp metal or glass knives, removed From the edge oF the kniFe either as individual sections
or as ribbons oF sections, and fl
oated in water (usually warmed) or in other fl
uids that are required by
the unique Features oF the specif c technique. ±or most applications in light microscopy, the sections
range in thickness From about 5 to 12
m
m (For paraFf
n embedded tissue) and From about 0.5 to 2.0
m
m
(For plastic embedded tissue). Special techniques may require sections up to 40 to 70
m
m in thickness. At
the opposite extreme oF the microscopic continuum, glass or diamond knives are used to cut extremely
thin sections For electron microscopy (EM, commonly called “TEM”). The thickness oF sections For
electron microscopy/transmission electron microscopy (EM/TEM) usually ranges From about 80 to
110 nm (10
−9
m).
AFter the sections are cut, and beFore they are mounted, it is routine to make sure the glass slides
are clean and to treat the slides so that the sections will not come oFF during the staining process. This
may be accomplished by either spreading a drop oF albumin on the slide or by adding a small amount
oF albumin (or gelatin) to the warm water bath in which the tissues are fl
oated. Celloidin can also be
used as an adhesive.
Staining
The goal oF staining tissue slices is to use substances to impart color to various components oF the
section, making these components available For study. In many cases, the stain will visualize a specif
c
component, such as Fat, neurof brils, or glycogen.
Stains are large molecules that are characterized by groups that absorb visible light (wavelengths
between 380 and 760 nm) and groups that permit the attachment oF the stain to the various chemical
elements oF the cell. The component oF the stain that is responsible For light absorption is a system oF
conjugated double bonds known as the
chromophore
. The “combining” portion oF the stain, which
may also Function as a solubilizing agent, is the
auxochrome
.
The simplest way that stains Function is to exploit the electrostatic interactions between the stain
molecules and components oF the cell; positive charges on cellular structures attract negatively charged
stain molecules and vice versa.
Basic dyes
carry positive charges and are, consequently, known as
cat-
ionic dyes
; they are attracted to negative charges within the tissue. Hematoxylin and toluidine blue are
commonly used basic (cationic) dyes. They stain nuclear DNA, cytoplasmic RNA, sulFonated polysac-
charides such as chondroitin sulFate, and polycarboxylic acids such as hyaluronic acid.
Acidic dyes
carry negative charges and are, consequently, known as
anionic dyes
; they are attracted to positive
charges within the tissue. Eosin Y is a commonly used acidic (anionic) dye. It stains many proteins
(and, thereFore, stains many structures within the cell), and acid dyes also stain extracellular structures
such as collagen.
There are literally hundreds oF substances that are used as stains or dyes, or that may be used to
impregnate tissues, and there are equally numerous methods or techniques that use these substances,
in various combinations, to visualize the components oF cells. Recognizing that even a brieF survey oF
the broad range oF methods is well beyond the scope oF this atlas; the Following Focuses on the major
stains used in this book.
Hematoxylin and eosin (H&E)
is, by Far, the most commonly used combination stain in basic
sciences For general histological preparations and in clinical medicine For pathological specimens. This
combination oF cationic and anionic dyes results in most constituents oF the cell (RNA, DNA, poly-
saccharides, and others) being stained various tones oF either blue or pink. This method also stains
extracellular collagen.
The
trichrome stains
are usually mixtures oF acidic dyes, each having its own ionization constant.
The Mallory trichrome stain, which is one oF the more common, is a mixture oF dyes used to dem-
onstrate connective tissue (collagen stains blue) and other cellular constituents (nuclei and cytoplasm
generally stain red), and blood cells (erythrocytes stain yellow). Trichrome stains are particularly
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