Iron stain )hemosiderin)
 Hemosiderin (storage iron granules) may be present in
areas of old hemorrhage or be deposited in tissues with
iron overload.
 Hemosiderosis is the term used if the iron does not
interfere with organ function; but hemochromatosis refers
to a condition of iron overload associated with organ
failure.
Perl's iron stain is the classic method for demonstrating iron in
tissues.
The section is treated with dilute hydrochloric acid to release ferric
ions from binding proteins. These ions then react with potassium
ferrocyanide to produce an insoluble blue compound (Prussian
blue reaction).
Mercurial fixatives seem to do a better job of preserving iron in
bone marrow than formalin.
Copper - RHODANINE METHOD
 Purpose: To identify copper deposits, such as in Wilson's Disease,
in the liver.
 Principle: It has been suggested that the rhodanine demonstrates
the protein to which the copper binds rather than the copper itself.
 Results:
Copper deposits: bright red to orange
Nuclei: blue
CALCIUM- VON KOSSA'S METHOD
 Purpose: Abnormal deposits of calcium may be found in any area
of the body. With the H&E stain, calcium appear deep blue-purple.
 Principle: Tissue sections are treated with silver nitrate solution,
the calcium is reduced by the strong light and replaced with silver
deposits, visualized as metallic silver.
 Results:
Calcium salts: black
Nuclei:
red
Cytoplasm:
pink
Urate Crystals - Gomori's Methenamine Silver
 Purpose: To demonstrate urate crystals.
 Principle: The urates take-up the silver, the silver is then
reduced to its metallic state.
 Results:
Urates: black
Background: green
 NOTE: Large calcium deposits may also stain black.
Melanin- Fontana-Masson Silver Method
 Purpose: To identify argentaffin granules and melanin. Melanin
is a non-lipid, non-hematogenous pigment. It is A brown-black
pigment present normally in the hair, skin, retina, iris and certain
parts of the CNS. Argentaffin granules are found in carcinoid
tumors.
 Principle: A positive argentaffin reaction means the
cells take-up silver and then reduce it to a visible
metallic state, without the aid of a reducing agent.
 Results:
Melanin, argentaffin cells: black
Nuclei: red
Fat - Oil Red O
 Purpose: To demonstrate fat or lipids in fresh tissue sections. Fat
occurring in an abnormal place, such as fatty emboli, and tumors
arising from fat cells (liposarcomas) can be differentiated from
other types of tumors.
 Principle: Staining with oil-soluble dyes is based on the greater
solubility of the dye in the lipoid substances than in the usual
hydroalcoholic dye solvents.
 Results:
Fat: red
Nuclei: blue
Fat - Sudan Black
 Purpose: For the demonstration of fat (using cryostat
sections).
 Principle: Sudan Black is slightly basic dye and will
combine with acidic groups in compound lipids, thus
staining phospholipids also.
 Results:
Fat: blue-black
Nuclei: red
Collagen - Masson's Trichrome Stain
 Purpose: Used to differentiate between collagen and
smooth muscle in tumors, and the increase of collagen in
diseases such as liver cirrhosis.
 Routine stain for liver and kidney biopsies.
 Principle: As the name implies, three dyes are
employed selectively staining muscle, collagen fibers,
fibrin, and erythrocytes.
 Results:
Nuclei: black
Cytoplasm, muscle, erythrocytes:
Collagen: blue
red
 Chronic active hepatitis
 Cerebral abscess
 Scleroderma with fibrosis
Reticular Fibers
- Gordon And Sweet's
Method
 Purpose: A silver impregnation technique that demonstrates reticular
fibers. Reticulum is abundant in liver, spleen, and kidney. Reticulum
forms characteristic patterns in relationship to certain tumor cells.
 Principle: The tissue is oxidized, then sensitized with the iron alum,
which is replaced with silver. The silver is reduced with formalin to its
visible metallic state.
 Results:
Reticular fibers: black
Nuclei:
red
Elastic Tissue Fibers - VAN GIESON Technique
 Purpose: This stain is useful in demonstrating atrophy of
elastic tissue in cases of emphysema, and the thinning and
loss of elastic fibers in arteriosclerosis, and other vascular
diseases.
 With increasing age, changes such as splitting and
fragmentation occur, these changes are most obvious in the
skin which becomes wrinkled and rather 'loose-fitting'.
 Principle: The tissue is stained with a regressive
hematoxylin, consisting of ferric chloride and iodine.
 Results:
Elastic fibers and nuclei: black
Collagen:
red
Other tissue elements: yellow