Visualization of Different Tissues Involved in
Endochondral Ossification With Alcian Blue
Hematoxylin and Orange G/Eosin Counterstain
Jessica R. Nowalk1 and Lisa M. Flick1,2*
1
Biomedical Materials Engineering Science and 2Division of Biology, Alfred University, Alfred, NY
Abstract
The histologic evaluation of endochondral ossification is
critical to the study of fracture healing, developmental biology, and comparative histology. A modification of Sayers’ alcian blue hematoxylin staining technique is described
in which a different counterstain is applied. Paraffin sections are stained in alcian blue hematoxylin for 30 min, and
then placed in orange G-eosin counterstain for 1 min, 30 s.
This method can differentiate cartilage, mature bone, and
immature bone found in various stages of endochondral
ossification and fracture callus. Increasing the hematoxylin
content to 0.5% produced a more striking contrast between
the bone and cartilaginous aspects of the healing fracture
callus. Inclusion of orange G (instead of acid fuchsin) in the
counterstain provides better demarcation of mature and immature bone. Alcian blue hematoxylin/orange G-eosin consistently stains cartilage blue, mature bone orange, and immature bone mauve and is particularly suited to the study of
endochondral ossification, fracture healing, and bone remodeling. (The J Histotechnol 31:19, 2008)
Submitted May 29, 2007; accepted with revisions October
1, 2007
Key words: alcian blue hematoxylin, endochondral ossification, fracture healing, orange G
Introduction
Endochondral ossification is the primary mechanism responsible for the embryonic development of long bones and
the resolution of long bone fractures (1). This process inThis work was supported by a grant from the New York State Foundation for Science, Technology and Innovation.
Address reprint requests to Lisa M. Flick, Division of Biology, Alfred
University, One Saxon Drive, Alfred, NY 14802.
E-mail: [email protected].
This article is approved for 1 contract hour through NSH, please visit
www.nsh.org for details.
The Journal of Histotechnology / Vol. 31, No. 1 / March 2008
volves the differentiation of chondrocytes that produce large
quantities of cartilage. Cartilage is later mineralized and
converted to new woven bone, which is further remodeled
over time, resulting in clinical union and restoration of torsional strength and stability (2). The study of bone development and factors affecting that process as well as experimental fracture models rely on histological examination to
evaluate the different tissue types, and therefore require a
staining method capable of discriminating between cartilage, immature bone, and mature bone. Alcian blue hematoxylin (ABH) was first described by Sayers et al. (3) as a
method to distinguish various types of growing and necrotic
bone. More recently, Sayers reported an improvement of the
original protocol that is faster and stains more intensely (4).
This paper describes a modification to the alcian blue hematoxylin technique, applying a new counterstain to enhance the differentiation of mature and immature bone.
Materials and Methods
Tissue Processing
Mouse tibia samples were collected 14 d after fracture.
Excess muscle tissue and internal fixation pins were removed from the fracture callus before fixation in 10% buffered neutral formalin for 24 h. Tissues were decalcified in
10% EDTA (pH 7.2) for 2 weeks. Decalcification occurred
with constant stirring and the 10% EDTA solution was
changed weekly. These were then processed through a series of 10 stations as follows: 70% ethanol (one station) for
2 h, 95% ethanol (two stations, 1 h each), 100% ethanol
(three stations, 1h each), xylene (two stations, 1 h each) and
two paraffin stations of 1 h each. Specimens were embedded
in paraffin and cut into 5-␮m sections.
Solutions:
1. 1% Acid alcohol: 1 mL of hydrochloric acid is added
to 99 mL 70% ethyl alcohol.
2. ABH: 2.5 g of hematoxylin (C.I. 75,290) is dissolved
by holding overnight in 350 mL of distilled water. The
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Figure 1. Experimentally produced tibial fractures from mice 14 d postfracture. Serial sections (original magnification ×40) from the same paraffin block
were stained with alcian blue hematoxylin/orange G eosin (A), Sayers’ ABH (B), Masson’s trichrome (C), and safranin-O/fast green (D). In (A), note the
presence of cartilage that stains blue, immature bone (mauve/gray, white arrows) and mature bone (orange, black arrows). (E) shows the alcian blue
hematoxylin/orange G eosin stained callus and trabecular bone at x400 original magnification. Arrows in (E) indicate osteoclasts (OCL) and osteoblasts
(OBL), respectively. Digital photomicrographs were taken using a standard Olympus compound microscope and an Olympus C-7070 camera.
next day, 25 g of aluminum ammonium sulfate and
0.25 g of sodium iodate are added and held overnight.
On day three, 150 mL of glycerol and 10 mL of glacial
acetic acid are added and stirred well. After the solution is filtered, 5 g of alcian blue (C.I. 74,240) is added
and stirred overnight. The solution is stored in a glass
bottle for 1 week before use to enhance solubility of
the alcian blue. The solution is stable for 3 months and
is stored in a dark cabinet.
3. 0.5% Ammonia water: 0.5 g of ammonium hydroxide
is added to 100 mL of distilled water.
4. Orange G-eosin counterstain: A 0.12% stock eosin
solution is made by adding 0.6 g of eosin Y (C.I.
45,380) to 450 mL 100% ethyl alcohol and 50 mL of
distilled water. The pH is adjusted to 4.6–5.0 by adding glacial acetic acid. A volume of 37 mL of 1%
phloxine B (C.I. 45,410), dye content 93%; and 16 mL
of 2% orange G (C.I. 16,230), dye content 94%; are
added to the eosin Y stock. This solution can be reused
for 1–2 weeks and the eosin stock solution is stable for
1 month.
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All chemicals are obtained from Sigma (St. Louis, MO)
unless otherwise stated.
Staining Procedure
1. Deparaffinize and hydrate slides.
2. Immerse slides in 1% acid alcohol for 30 s, then
drain well.
3. Stain slides in ABH for 30 min at room temperature.
4. Wash slides well in distilled water.
5. Differentiate in 1% acid alcohol for 2–3 s.
6. Wash well in distilled water.
7. “Blue” in 0.5% ammonia water for 15 s.
8. Wash well in distilled water.
9. Immerse slides in 95% alcohol for 1 min.
10. Drain slides well and place in the orange G-eosin
stain for 1 min and 30 s.
11. Rinse slides in 3 changes of 95% alcohol, leaving the
slides in the last alcohol for 3 min.
Histologic Evaluation of Endochondral Ossification Tissues / Nowalk and Flick
12. Dehydrate, clear in xylene, and mount coverslips using synthetic resin.
Results
Mid-diaphyseal long bone fractures heal via endochondral ossification, which involves chondrocyte differentiation and the mineralization of new woven bone (1). Fourteen days after fracture, the callus is characterized by the
presence of cartilage, mature bone, and woven (immature)
bone (1). Various stains were tested for their ability to discriminate cartilage and bone, including ABH/orange Geosin, Masson’s trichrome, safranin-O/fast green, and Sayers’ ABH (Figure 1) (4). Alcian blue hematoxylin/orange
G-eosin was the only stain that could distinguish all of these
differentiation states. Cartilage stains blue, mature bone appears orange (black arrows), and immature bone mauve/
gray (white arrows, Figure 1A). Good contrast is also seen
between the muscle tissue (red), fibrous connective tissue
(violet), and erythrocytes (bright red, Figure 1A). Late stage
fracture callus is characterized by the presence of both osteoclasts and osteoblasts, both of which are visible under
high magnification when stained with alcian blue hematoxylin/orange G-eosin (Figure 1E). Bone cells (including
osteoblasts and osteoclasts) are pink with purple nuclei.
Discussion
The stain described here was applied to samples of partially healed long bones in mice although the applications
are far more diverse, including developmental biology studies, comparative histology, etc. The ABH/orange G eosin
stain was found to be extremely useful since neither Sayers’
ABH, safranin-O/fast green, nor Masson’s trichrome were
able to differentiate between cartilage, new bone, and old
bone. Safranin-O/fast green staining of fracture callus did
indicate a difference between bone (light blue–green) and
cartilage (red, see Figure 1D); however, it did not allow for
the identification of mature vs. immature bone. Sayers’
ABH stain also distinguishes cartilage (blue) from immature
The Journal of Histotechnology / Vol. 31, No. 1 / March 2008
and mature bone (various shades of pink, Figure 1B). Two
problems noted with Sayers’ ABH were consistency and
contrast; both immature and mature bone were difficult to
distinguish and did not stain consistently. The modification
presented here overcomes both of these problems, staining
immature bone mauve/gray and mature bone orange with
great contrast provided by the cartilage, which stains blue.
Additional advantages to the new adaptation include a
greater concentration of hematoxylin (0.5% vs. 0.15% for
the Sayers method) and a simpler protocol for the production of ABH. This stain is also very durable; color intensity
is maintained in mounted sections 1 year later. Although the
best method for distinguishing mature bone, immature bone,
and cartilage remains undecalcified plastic-embedded histology, the method described here is more versatile, allowing other analyses and methods (such as cytochemical staining or immunohistochemistry) to be used with the same
tissue block. Although it is likely that the alcian blue hematoxylin/orange G eosin method would work with other
fixative or decalcification agents, this has not been evaluated because 10% neutral buffered formalin and ethylene
diamine tetraacetic acid represent the most common chemicals used for these applications.
Aknowledgement
We thank Jennifer L. Harvey for her expert technical
advice.
References
1. Einhorn TA: The cell and molecular biology of fracture healing. Clin Orthop 355 Suppl:S7–21, 1998
2. Cruess RL, Dumont J: Fracture healing. Can J Surg 18:403–
413, 1975
3. Sayers DCJ: A general staining technique for the demonstration of new bone. J Sci Technol 17:14–15, 1973
4. Sayers DCJ, Volpin G, Bentley G: The demonstration of bone
and cartilage remodelling using alcian blue and hematoxylin.
Stain Technol 63:59–63, 1988
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