a. Gomori’s Chromaffin Stain Results: Chromaffin granules—purplish red Only alpha cells of pancreatic islets, some cells of anterior pituitary and granules of neutrophils and myelocytes
1. Deparaffinize the sections through two changes each
of xylene, absolute alcohol, and 95% alcohol.
2. Rinse well in distilled water.
3. Place in the following bleaching solution for 5 minutes
4. Wash well in running tap water and distilled water, and
Hematoxylin, a natural dye, is the most important staining
reagent used in histologic work.Used alone, it has little affinity
for tissue; but in combination with (mordants) aluminum,
iron, chromium, copper or tungsten salts, it is a powerful
nuclear stain and a chromatin stain. The active coloring
agent, hematein, is formed by the oxidation of hematoxylin.
This process, known as “ripening”, takes several days or
weeks unless it is hastened by the addition of an oxidizing
agent, such as mercuric oxide, hydrogen peroxide, potassium
permanganate, sodium perborate or sodium iodate.
Most commonly used hematoxylins are used in
combination with aluminum in the form of alum. Various
formulations used go by the following names, e.g. Harris,
Mayer, Delafield, Ehrlich, Bullard and Bohmer.
There are two methods of staining when hematoxylin is
used: progressive and regressive.
Progressive: The hematoxylin contains an excess of
aluminum salts or acid, thus increasing the selectivity
for nuclei. After staining with hematoxylin, the slides are
washed well in water and counterstained.
Regressive: This is accomplished by overstaining in a
relatively neutral solution of hematoxylin, then removing
the stain from the other constituents with acid alcohol or
Commonly Employed Hematoxylins
Ammonium or potassium alum 100.0 g
Dissolve the hematoxylin in the alcohol, the alum
in water by the aid of heat. Remove from heat and mix
the two solutions. Bring to a boil as rapidly as possible.
Remove from the heat and add the mercuric oxide slowly.
Reheat until it becomes dark purple, remove from flame
immediately and plunge the vessel into a basin of cold
water until cool. The stain is ready for use as soon as it
cools. Addition of 2 to 4 cc of glacial acetic acid per 100
cc of solution increases the precision of the nuclear stain.
Ammonium or potassium alum 50 g
Dissolve the hematoxylin in water, using gentle heat if
necessary. Then add the sodium iodate, then the alum.
Shake until the alum is dissolved, then add the citric acid,
and finally the chloral hydrate. The final color of the stain
is reddish-violet. Stain will keep well.
Counterstains for Hematoxylin Stains
Stock 1% Aqueous Eosin Solution
Stock 1% Alcoholic Eosin Solution
Eosin, 1% stock solution 1 part
If deeper shade of red is desired in staining, add 0.5 cc of
glacial acetic acid to each 100 cc of stain.
Routine Hematoxylin and Eosin Stain
Fixation: May be used after any fixation.
¾ Cut paraffin sections at 6 microns.
Hydrochloric acid, concentrated 10 cc
Strong ammonia water 2 to 3 cc
4. Saturated lithium carbonate solution
6. Lugol’s solution (Weigert’s modification)
8. Sodium thiosulfate solution (Hypo)
796 Concise Book of Medical Laboratory Technology: Methods and Interpretations Staining Procedure
1. Xylene, absolute alcohol, 95% alcohol.
2. If sections are Zenker’s fixed, treat with either Lugol’s
solution or 1% alcoholic iodine solution 10–15
minutes. Wash with tap water. Treat with 5% sodium
thiosulfate 5 minutes. Wash well with tap water.
3. Harris’s hematoxylin for 15 minutes.
5. Differentiate in acid alcohol—3 to 10 quick dips.
Check the differentiation with the microscope—
nuclei should be distinct and the background very
6. Wash in tap water very briefly.
7. Dip in ammonia water (for 10–20 seconds) saturated
lithium carbonate solution until sections are bright
8. Wash in running tap water for 10–20 minutes.
9. Stain with eosin for 15 seconds to 2 minutes
depending on the age of the eosin and the depth of
11. Absolute alcohol—at least two changes.
a. Mallory’s Phosphotungstic Acid Hematoxylin Stain
b. Mallory’s Phosphomolybdic Acid Hematoxylin Stain
c. Heidenhain’s Iron Hematoxylin Stain
Chromatin, nucleoli, mitochondria, and parts of striated
muscle fibers are stained black. Other tissue elements are
stained by contrast stain used. Demonstrates amoeba.
d. Mallory’s Aniline Blue Collagen Stain
Ground substance of cartilage, mucin and amyloid—
Erythrocytes and myelin—yellow
Elastic fibrils—pale pink, pale yellow or unstained.
e. Lee-Brown’s Modification of Mallory’s Aniline Blue Stain
Glomerular basement membrane of kidney—deep blue.
f. Van Gieson’s Stain for Collagen Fibers
g. Barbeito-Lopez Trichrome Stain
h. Gomori’s One-Step Trichrome Stain
Striations of muscle are easily demonstrable.
i. Heidenhain’s Aniline Blue Stain
Chromatin, osteocytes and neuroglia—red
j. Gallego’s Iron Fuchsin Stain
Collagen and reticulum—deep blue
Muscle—greenish to orange yellow
Calcium—reddish to purple brown
k. Weigert’s Resorcin-Fuchsin Elastic Stain
Elastic fibers—blue-black to black
l. Gomori’s Aldehyde Fuchsin Stain
Elastic fibers and mucin—deep blue
Beta cells of pancreas—deep blue
o. Koneff’s Stain for Bone and Cartilage
Hyaline cartilage and osteoid—blue
Bone—bright red to reddish brown
Matrix of vesicular zone of epiphyseal disc and remnants
Stains for Cytoplasmic Granules
Chromaffin granules—purplish red
Only alpha cells of pancreatic islets, some cells of anterior
pituitary and granules of neutrophils and myelocytes
b. Acid Fuchsin Aniline Blue Method for Pituitary Granules
Acidophil granules—orange vermillion
Basophil granules—deep cobalt blue
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