Review

Journal of Histochemistry & Cytochemistry 59(3) 252­–257

© The Author(s) 2011
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DOI: 10.1369/0022155410397760
http://jhc.sagepub.com

Viewing Hyaluronan: Imaging Contributes to Imagining New

Roles for This Amazing Matrix Polymer

Carol A. de la Motte and Judith A. Drazba

Department of Pathobiology (CAM) and Imaging Core (JAD), Cleveland Clinic Lerner Research Institute, Cleveland, Ohio.

Summary
Hyaluronan (HA) is an ubiquitous extracellular matrix polymer that plays many roles in health and disease. The ability to
view the spatial and temporal expression of HA in tissues and on/in cells has provided researchers with insights into the
tremendously diverse biological processes in which HA is involved. Biochemical extraction, quantity, and size measurement
of HA can tell part of the story, but these techniques are incomplete in placing HA at the scene of a biological event and
determining which other molecules are likely to be cooperating. HA, however, is not immunogenic, so preparing antibodies
for histochemistry is problematic. Fortunately, a probe for HA was devised based on the HA binding region of aggrecan,
and today this probe is commercially available and very useful for histochemistry. This article discusses the conditions and
considerations that the authors’ lab and others have developed for optimal HA staining in many tissues and cell types.
(J Histochem Cytochem 59:252–257, 2011)

Keywords
hyaluronan, confocal imaging, fluorescent staining, hyaluronan binding probe, colon, endothelial cells

Despite its uncomplicated chemistry, or perhaps as some
investigators believe because of it, hyaluronan (HA) is cen-
tral to many complex biological functions in chordates,
including promoting embryogenesis (Camenisch et al.
2000; Shukla et al. 2010), acting as part of the structural
extracellular matrix (Timmons et al. 2010; Alaniz et al.
2009), and signaling innate host defense mechanisms
(Forteza et al. 2001; Noble and Jiang 2006). HA is a chemi-
cally simple, non-branching polymer of repeating units of
N-acetyl-glucosamine and glucuronic acid. In normal tis-
sue, HA is usually present as very large molecules (106−107
Da), although during inflammatory processes, the polymers
can be cleaved to fragments of lower molecular weight,
which take on new cell signaling roles (Stern et al. 2006).
The ability to see the location, time of appearance, and
structure of HA in tissues and on/in cells has provided
researchers with insights into the tremendously diverse bio-
logical processes in which HA is involved. Biochemical
extraction, quantity, and size measurement of HA can tell part
of the story, but these techniques are incomplete in placing
HA at the scene of a biological event and determining which
other molecules are likely to be cooperating at the same time.
Two examples emphasize this point well (de la Motte et al.
1999; de la Motte, Hascall, Drazba, Bandyopadhyay, et al.
2003; Kessler et al. 2008). When comparing cultures of intes-
tinal smooth muscle cells that were untreated to those acti-
vated with a viral mimic, poly I:C, a 10-fold or greater
increase in HA-mediated leukocyte adhesion was consis-
tently observed. Yet only a 2.5-fold increase in cell-associated
HA was measured (de la Motte et al. 1999). Through the use
of imaging, we gained the insight that much of the induced
HA formed a cable structure that could bind non-activated
Received for publication November 15, 2010; accepted December 23,
2010
Corresponding Author:
Carol de la Motte, PhD, Department of Pathobiology–NC2, Cleveland
Clinic Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH
44195.
E-mail: delamoc@ccf.org
Imagining New Roles for This Amazing Matrix Polymer
leukocytes, whereas the cell glycocalyx that excludes parti-
cles was not leukocyte adhesive. This observation led to the
investigation of whether incorporation of binding proteins
into the HA matrix—specifically, the heavy chains of inter-
alpha-trypsin inhibitor (IαI; de la Motte, Hascall, Drazba,
Bandyopadhyay, et al. 2003) and versican (de la Motte,
Hascall, Drazba, Strong 2003; Potter-Perigo et al. 2010)—is
important for cable formation and function. Blocking the
incorporation of either of these two proteins inhibits leuko-
cyte adhesive function (de la Motte, Hascall, Drazba,
Bandyopadhyay, et al. 2003; Potter-Perigo et al. 2010).
However, blocking IαI binding to HA results in loss of cable
structure formation, yet coat structures still form. IαI heavy
chains are now known to increase the adhesiveness of HA
(Zhuo et al. 2006) for leukocytes, which suggests that a major
reason the pericellular matrix does not interact with leuko-
cytes while cables structures do is based on the incorporation
of IαI heavy chains. Although versican is incorporated in
both pericellular coat and cable HA, we envision that versi-
can, as presented on the cable structure, is more accessible
for helping to facilitate leukocyte adhesion.
In vivo, similar shifts in HA deposition have been
observed in intestinal tissue that would not be reflected in
HA measurements. Mouse large intestine normally contains
organized HA in the epithelial layer, likely to aid water
absorption and maintain flexibility of the organ. During
intestinal inflammation, however, the epithelial HA disap-
pears, and new deposition in subepithelial tissue occurs
(Kessler et al. 2008), although total HA content in the intes-
tinal wall does not change appreciably. Once again, HA
was found to associate with binding proteins, the heavy
chains of IαI, in the inflammatory context. Figure 1 is an
example of how tissue imaging gave many clues to important
inflammation-associated changes in HA that biochemical
techniques would not have uncovered. Panels 1a and 1c show
different magnifications of cross sections through a normal
mouse colon, where organized HA surrounds the epithelial-
lined (“e”) intestinal crypts. During inflammation (panels 1b
and 1d), the crypts drop out, and differently structured HA is
densely deposited beneath the damaged epithelial layer. In
addition, HA is deposited in the submucosal space (“s”),
which greatly expands during inflammation. Higher magnifi-
cation of the submucosal tissue shows a change in HA depo-
sition as well as IαI binding protein incorporation during
inflammation (typified by the cellular infiltrate, as seen by
the increase in the number of blue-stained nuclei).
Because HA has a very simple, conserved composition
and is ubiquitously expressed in all animals that have a
developed immune response, HA is not immunogenic.
Therefore, there are no antibodies that specifically recog-
nize HA, and traditional immunohistochemical methods of
detection of HA are not possible. Fortunately, a very spe-
cific and tightly binding protein, the hyaluronan binding
protein (HABP), which is composed of the HA binding
253
domain with the link module from aggrecan, was isolated
(Hascall and Heinegård 1974; Tengblad 1979) and adapted
as an HA probe (Ripellino et al. 1985). HABP is now widely
employed for specific detection of HA.
The HABP probe has been successfully used for detection
of HA in cell cultures (live [Rilla et al. 2008; Evanko et al.
2009] and fixed [de la Motte, Hascall, Drazba, Bandyopadhyay,
et al. 2003; Kessler et al. 2008; Twarock et al. 2010; Selbi et al.
2006]), as well as in tissue sections (fixed, paraffin-embedded
sections as well as cryopreserved, OCT-embedded sections;
Noble and Jiang 2006; Stern et al. 2006; McDonald et al. 2008;
Auvinen et al. 2005; Lin et al. 1997). Fortunately, HABP cou-
pled to biotin can be purchased from many sources today. The
addition of biotin allows detection with common secondary
streptavidin reagents that are fluorescent or colorimetric.
Radiolabeled streptavidin (e.g., 125Iodine-streptavidin) is also
available for relative quantification of HA in the cell matrix (de
la Motte, Hascall, Drazba, Bandyopadhyay, et al. 2003).
Fixation with alcohols or aldehydes does not appear to affect
HABP binding to HA, but the choice of method does affect
condensation of the matrix and may alter detection of
HA-associated proteins in tissue using specific antibodies. One
possible caution investigators must be mindful of is that HA
that is highly bound to specific proteins, such as versican,
aggrecan, or TSG-6, may be underreported by the HABP.
Prior to the use of HABP, a specific histochemical reac-
tion was used for detection of HA in tissues. In fact, nearly 60
years ago, attempts to determine which reactions actually
stained components of mucopolysaccharides, including HA,
using histochemical procedures were being reported (Davies
1952). Because the components associated with HA were not
fully appreciated at the time, some methods (e.g., periodic
acid Schiff method) gave varied results, and the conclusions
reported were often imperfect (Zugibe 1962). Staining with
the cationic dye Alcian blue became the method of choice for
glycosaminoglycan detection, and differential staining of sul-
fated glycosaminoglycans and HA was reported (Hodson and
Prout 1968). The specificity of differential pH staining has
been questioned in numerous histological studies (Lin et al.
1997; Derby and Pintar 1978).
Then and now, a critical validation of whether a stained
sample contains HA is achieved by abrogating staining with
specific hyaluronidase treatment, but not with specific chon-
droitinase treatment, in replicate samples (Derby and Pintar
1978). Streptomyces hyaluronidase is considered the most spe-
cific HA-degrading enzyme, and chondroitinase ABC used at
pH 8.6 is the most specific for chondroitin sulfate.
The Process
Fixation
As mentioned above, cell cultures and excised tissue may
be preserved in either alcohol-based or aldehyde-based
254 de la Motte and Drazba

Figure 1. Changes in hyaluronan (HA; green) arrangement and distribution are among the most dramatic alterations in the colon during
induction of inflammation. (a, c) Untreated control; (b, d) induced colitis. High-magnification images (c, d) reveal that heavy chains of
inter-alpha-trypsin inhibitor (IαI; red) are heavily co-localized (yellow) with HA in the submucosa (star) in the inflamed intestine (d) but
little in the mucosa (arrowhead) or in the normal control section (c). HA was detected with biotinylated hyaluronan binding protein and
Alexa 488–streptavidin and imaged with a Leica confocal microscope. e, epithelial layer; s, submucosa; m, external muscle layer.

fixatives and successfully used for hyaluronan detection.
However, the patterns of staining are specific to the fixa-
tive, so direct comparisons between tissues fixed under
different conditions are not possible (Lin et al. 1997).
Evanko et al. (2009) have recently tested and compared the
common in vitro fixation methods used in HA studies.
Unlike the other glycosaminoglycans that have covalently
linked protein components, HA is not necessarily strongly
integrated into the matrix, and washout during processing is
possible. Cetylpyridinium chloride (CPC) precipitation of
HA in tissue results in superior retention in loose connec-
tive tissue (Ripellino et al. 1985; Lütjen-Drecoll et al. 1990;
Nishikawa et al. 1996). In relatively dense matrix where
HA is heavily linked with other proteins such as CD44,
aggrecan, and versican, CPC is not generally used. In addi-
tion, CPC is autofluorescent and therefore would interfere
with specific fluorescent detection methods.
Our lab methods rely primarily on alcohol-based fixa-
tion procedures, with excellent results predominating.
Recently, Evanko et al. (2009) have demonstrated in com-
parison assays that acid-formalin-ethanol fixative (3.7%
formaldehyde-PBS, 70% ethanol, and 5% glacial acetic
acid, all v/v) described previously (Lin et al. 1997) is supe-
rior in preserving the very fine HA matrix structures and
Imagining New Roles for This Amazing Matrix Polymer
resulted in less HA washout than formalin fixation. For
cells grown in monolayer culture, cold (−20C) 100% meth-
anol fixation for 10 min followed by air-drying generally
works well for preserving the lengthy HA cable structures.
Using alcohol fixatives as opposed to aldehydes has the
added benefit that antigen retrieval—sometimes required
because aldehydes can crosslink proteins and change the
antigenic structure—can be avoided when antibody co-
staining in the same preparation.
For colon tissue preservation of HA, we obtain excellent
results by fixation with Histochoice, a product that contains
a low concentration of the aldehyde, glyoxal. Freshly dis-
sected tissue submerged in Histochoice (>10 volumes
Histochoice/1 volume of wet tissue sample) may remain for
1 and 7 days at room temperature before processing and
paraffin embedding. However, Histochoice may not be
optimal for preservation of HA in all tissues, and most
investigators recommend directly comparing fixatives for
specific tissues and species initially before conducting large
experiments. Formalin fixed or fresh-frozen tissues also
successfully stain for HA. In our application, formalin fixa-
tion results in higher background autofluorescence that
interferes with our confocal fluorescence microscopy
results, whereas the fresh-frozen method compromises tis-
sue morphology. The choice of preservation method can
also be dependent on which antibodies are being used for
co-detection of molecules in addition to HA.
Staining
Commercial preparations of HABP form a very stable
complex with HA. As mentioned, the addition of biotin
allows easy detection of the probe. (There is widespread,
successful use of the commercial reagents from Associates
of Cape Cod, Inc. [East Falmouth, MA] and Calbiochem
[La Jolla, CA].) Most published accounts use the HABP
probe at a concentration of 1 to 5 µg/ml (de la Motte,
Hascall, Drazba, Bandyopadhyay, et al. 2003; Rilla et al.
2008; Evanko et al. 2009), generally suspended in a saline
solution (e.g., PBS, Hank’s balanced salt solution) con-
taining protein (usually non-immune serum or albumin) to
reduce nonspecific attachment of the binding probe to tis-
sue or the slide. We and other investigators have found that
binding of the HABP probe to HA does not seem to occur
as rapidly as antigen-antibody reactions. Optimal staining
is achieved after 12- to 16-hr incubation times. For the HA
labeling step, the sample is incubated at 4C to prevent
bacterial growth and in a humidified chamber (i.e., a
sealed plastic container with damp paper towels in the bot-
tom) to prevent drying. After incubation, slides or cover
slips carrying the samples are washed and reincubated
(30 min, ambient temperature) with streptavidin that is
conjugated to a fluorescent label (e.g., Alexa 488
Streptavidin [green] or Alexa 568 Streptavidin [red], both
255
from Molecular Probes [Eugene, OR], used at 1:500 dilution
in a saline solution) for fluorescence microscopy detec-
tion. Alternatively, using streptavidin linked to an enzyme
label (e.g., peroxidase) that is further processed for a colo-
rimetric reaction can also yield clear HA detection results
(Auvinen et al. 2005).
Imaging
Hyaluronan can form huge (at least on a cellular scale)
structures in tissue and in the matrix of cell cultures (de la
Motte, Hascall, Drazba, Bandyopadhyay, et al. 2003). The
HA structures are three-dimensional, even in fixed cells or
tissue where the matrix has been condensed. Observing
stained HA on a traditional microscope, whether brightfield
or fluorescence, is possible with continuous focal plane
adjustment, but the observed images all contain out-of-focus
light from adjacent planes of focus, and capturing a clear
image of the whole three-dimensional molecule is impos-
sible. To avoid this limitation, we use a confocal micro-
scope that allows multiple planes through the sample to be
captured—each in focus—and then viewed as a series, inte-
grated into a single image, or used to create three-dimen-
sional reconstructions that can be examined from all sides.
Figure 2a illustrates HA structures produced on the surface
of tumor necrosis factor (TNF)–α–activated endothelial
cells and the important role of confocal microscopy in visu-
alizing the extensive matrix. The single planes capture dif-
ferent information that can be integrated into an overlay
that best represents the whole cable structure. When fluo-
rescently tagged antibodies are used to label other proteins
in conjunction with HABP labeling, the multicolor confocal
images provide a wealth of information about the structural
associations of the tagged molecules (Figure 2b).
HA can also be tagged and imaged in live-cell cultures.
This can be achieved either by (1) adding a fluorescently
conjugated protein that binds to HA, such as Neurocan–
green fluorescent protein (GFP) fusion protein (Zhang et al.
2004) or fluoresceinated HABP (Ripellino et al. 1985), both
used successfully to image real-time HA changes in live
cultures, or (2) adding commercially available biotinylated
HABP directly to the culture medium, followed by a gentle
wash, incubation with a fluorescently tagged streptavidin,
and another wash at set times of interest. The live cells
are best imaged using a spinning disk confocal or a point-
scanning confocal with a resonant scanner that can collect
stacks of images very rapidly at regular intervals over time.
Such time-lapse imaging allows for the study of factors that
affect HA formation and that influence its cellular interac-
tions while the events are occurring.
HA is so much more than a space-filling matrix mole-
cule, although that role is also important. Increasingly,
investigators are appreciating that HA is a cue that activates
or inhibits physiological as well as disease responses (Stern
256 de la Motte and Drazba

Figure 2. Confocal microscopy aids in visualizing different cellular configurations of the hyaluronan (HA; green) matrix. Panel a shows
representative serial slices and the projection of the 16-micron z-stack from tumor necrosis factor (TNF)–α–activated microvessel
endothelial cells to which leukocytes have bound. Different structural features are evident in different focal planes, whether close to the
endothelial surface or wrapped around the leukocytes above the monolayer. The features are more evident in panel b, the same z-series
in which cell location is identified by nucleus staining (DAPI stain—blue), and leukocytes are labeled with CD44 antibody (red). Imaging
has led to the understanding that activated endothelial monolayers produce an HA matrix that contains different configurations: (1)
cable-like (arrowhead) structures that bind leukocytes (red) and (2) cell surface coat-like (star) arrays that are not leukocyte adhesive.

et al. 2006). In addition to changes in quantity, HA’s spatial
and temporal expression, as well as the proteins with which
it associates, all affect the function. The ability to specifi-
cally label and image HA has aided many groups in defin-
ing these parameters and contributed to understanding the
polymer’s role in normal physiology and development, as
well as during disease.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the authorship and/or publication of this article.
Funding
The research in this article was supported by National Institutes of
Health grants DK58867 and DK069854.
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