Iozzo Renato V. Proteoglycan Protocols
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Localization of FGF-specific HSPGs 541
2. Transfected COS-7 cells are grown in selection medium in T175 flasks. Once confluent,
the cells are suspended by trypsinization, and plated in 850-cm
2
roller bottles (1 flask per
roller bottle) in 100 mL of culture medium. The bottles are gassed with 5% CO
2
/air
mixture, capped tightly, and placed in a 37°C roller bottle apparatus. Once the roller
bottle is 50–60% confluent, the medium is replaced daily. The conditioned medium is
saved, snap-frozen in liquid nitrogen, and stored at –70°C.
3. The anti-AP antibody affinity column is stored in the cold room in 0.02% NaN
3
as a
preservative. Before using the column, wash with 5–10 bed volumes of PBS.
4. Conditioned medium from the transfected COS cells is filtered to remove cell debris and 250
mL are applied to a 1-mL anti-AP antibody affinity column. The conditioned medium
typically contains 1–10 µg/mL FR1c-AP.
5. After washing the column with 5 column volumes of PBS, FR1c-AP is eluted from the
column with 3.6 mL of AP elution buffer. Fractions (0.3 mL) of the eluate are collected
directly into an equal volume of neutralization buffer. Immediate neutralization is important
to preserve the activity of AP and probably also ligand-binding activity. Following elution,
the eluate is dialyzed against PBS.
6. The concentration of purified FR1c-AP is determined is several ways. It is quantified in an
AP assay using p-nitrophenylphosphate as a substrate and comparing the activity with
human placental alkaline phosphatase as standard. Aliquots containing FR1-AP are mixed
with an equal volume of 2× AP assay substrate and incubated at 37°C for 10, 20, and
30 min before reading absorbance at A
405
. Second, its purity is assessed using SDS-PAGE
gels stained with Coomassie blue and concentration is estimated using human PLAP as a
standard. Finally, once the preparations are shown to be pure, protein is determined using
a BCA protein assay (Pierce).
3.3. Preparation of Tissue Sections
The binding and localization procedure can be performed using either frozen or
paraffin-embedded tissue sections. When fresh-frozen tissues are being examined, the
tissue should be embedded in OCT or equivalent compound (see Note 2).
1. (Frozen sections) 4-µm sections are thaw-mounted on charged slides (e.g., Fisher Plus
slides) and immediately dipped in 70% ethanol and then fixed in 4% paraformaldehyde in
PBS at room temperature. The fixation procedure is crucial for reproducible FR1-AP bind-
ing of high intensity.
(Paraffin-embedded sections) Formalin-fixed and paraffin-embedded tissues are also cut at
4 µm and mounted on charged slides. Paraffin sections are rehydrated according to standard
histology technique by melting and exposure to xylene and a series of graded alcohols.
2. When bound protein probes are to be detected by fluorescence microscopy, autofluorescence
is reduced by treating the tissues with sodium borohydride (0.5 mg/mL; prepared in 4°C
double-distilled H
2
O, and then warmed to room temperature). Following a 10 min incubation,
the slides are washed in PBS and incubated with glycine (0.1 M) in PBS overnight at 4°C.
3. To reduce nonspecific binding, the sections are blocked before addition of antibody (for
3G10 staining) or before growth factor incubation with either blocking solution.
4. The staining and in situ binding reactions can be carried out in a humidified chamber. We
have also found vertical slide staining racks (Sequenza System) to be very useful, especially
when staining large numbers of slides. The cover plates used in the Sequenza racks create a
capillary space above the tissue section, allowing for even and consistent antibody/growth
factor exposure. The staining intensities are somewhat reduced when using this system,
compared to slide incubation in the humid chamber.
542 Friedl et al.
3.4. Localization of Heparan Sulfate
The detection of heparan sulfate relies on cleavage of the glycosaminoglycan by a
highly specific heparitinase. Preparations of this enzyme are commercially available,
and different forms recognize different sulfation or sugar sites in the heparan sulfate
chain. Because these enzymes are lyases rather than hydrolases, they generate a novel
unsaturated glucuronate residue at the nonreducing terminus of the chain fragment
that remains attached to the core protein. This unsaturated glucuronate is antigenic,
and antibodies (e.g., mAb 3G10) for its detection are commercially available. Thus,
detection of the heparan sulfate in the tissue requires only a single clip by the
heparitinase somewhere in the length of the chain. Antibody 3G10 and the immuno-
histochemistry method have been developed by David and co-workers (20):
1. The tissue sections (frozen sections or rehydrated paraffin sections) are treated with
heparitinase. We use generic heparitinase at 10 mIU/mL in heparitinase buffer for 4 h at
37°C, replenishing the enzyme after 2 h.
2. After blocking for 30 min, the sections are incubated with anti-delta-HS antibody 3G10 at
2 µg/mL for 60 min. After washing, the bound antibody is detected by a fluorescent sec-
ondary antibody (frozen sections) or an HRP-conjugated secondary antibody (paraffin
sections).
3. An important control is to omit the heparitinase digestion step. Sections stained without this
prior step lack the unsaturated glucuronate antigen, and antibody staining is typically seen to
be absent.
3.5. Binding of FGF and FR1c-AP to Tissue Sections
1. After blocking, the tissue sections are incubated with FGF (e.g., FGF-2 at 3–10 nM) in
blocking solution for 60 min.
2. Unbound FGF is removed by washing 5 times in TBS. After washing, continue to step 3
to detect bound FGF by antibody staining, or to step 5 to carry out FR1c-AP binding to
the FGF/HS complexes.
3. After washing in TBS to remove unbound growth factor, the sections are incubated with
anti-FGF-2 antibody, or with anti-biotin antibody for 60 min.
4. Bound FGF is detected with fluorescent (frozen) or HRP-conjugated secondary antibody or
avidin (for biotinylated FGF).
5. Alternatively, rather than detect the bound FGF ligand with antibody, sections containing
bound, exogenous FGF are incubated with FR1c-AP at 10–30 nM for 60 min at room
temperature.
6. Unbound FR1c-AP is removed by 5 washes in TBS.
7. Bound FR1c-AP is detected with anti-placental alkaline phosphatase antibody. Antibody is
detected as described below.
3.6. Important Binding Controls
The purpose of the binding assays is to detect (1) HSPGs capable of binding FGF
ligand and (2) HSPGs capable of forming the complete FGF signaling complex. There-
fore, the controls have to prove that the FGF-binding sites are HSPGs and that soluble
receptor binding is FGF-dependent. The HS identity of the FGF-binding sites can be
demonstrated by treating the tissue sections with heparitinase before the FGF binding
step. Bacterial heparitinase has a high degree of substrate specificity and will not
Localization of FGF-specific HSPGs 543
degrade other glycosaminoglycans such as chondroitin sulfate or hyaluronate. Best
results are obtained by treating the sections for 4 h with a mixture of generic
heparitinase and heparitinase II, both at 10 mIU/mL in heparitinase buffer, for 4 h at
37°C. The enzyme is replenished after 2 h. Using the combination of the two enzymes
with different heparan sulfate-specific scission sites results in more efficient signal
reduction. FGF dependence of soluble receptor binding is shown by omitting the FGF
incubation step.
3.7. Detection Systems
For antibody detection by fluorescence microscopy, we recommend secondary
antibodies conjugated with Alexa dyes by Molecular Probes (Eugene, OR). The Alexa
fluorochromes are available for excitation and emission wavelength compatible with
commonly used filters, deliver a signal of high intensity, and are very resistant to
fading. For viewing with a regular bright-field microscope and for paraffin sections,
the DAKO detection kits have been used by us with good success. The polymer-based
Envision-Plus® or the biotin-avidin-based LSAB-Plus detection systems combine
high sensitivity with low background.
4. Notes
1. The combined application of these in situ assays has allowed us to distinguish HSPGs that
bind FGF-2 from those that bind FGF-7 (18), and to classify HSPGs as promoters and inhibi-
tors of FGF-2 signaling complex formation (19). In human skin, an organ governed by sig-
naling of different members of the FGF family, distinct differences are detected between
keratinocyte surface HSPGs (likely members of the syndecan and glypican families of
HSPGs) and epidermal basement membrane HSPGs (predominantly perlecan). Specifically,
we find that keratinocyte surface HSPGs bind both FGF-2 and FGF-7, while epidermal base-
ment membrane HSPGs bind FGF-2 but not FGF-7 (see Fig. 2B,C). Similarly, distinct dif-
ferences between cell surface and basement membrane HSPGs are detected with regard to
soluble FGF RTK binding. Epidermal surface HSPGs immobilize FR1c-AP, while epider-
mal basement membrane HSPGs failed to so (see Fig. 2E), despite the fact that HSPGs in this
extracellular location bind ample amounts of FGF-2 (see Fig. 2C). These in situ techniques
enable one to examine HSPG location and function in model systems where HSPGs have
been found to be dynamically regulated and where FGFs play important roles, such as during
development and in pathological conditions. The applicability of the assays to paraffin-
embedded archival tissues opens the door to retrospective analysis of pathological specimens.
2. A few limitations of the in situ binding assays have to be emphasized. The assays do not
allow the clear assignment of a certain HSPG species to FGF binding or signaling; they
localize HS-binding activities and, when combined with immunohistochemical detection of
HSPG core proteins, the HS may be shown to co-localize with a specific core protein. Also,
it cannot be ruled out that endogenously produced HS-binding ligands may mask some of
the binding sites on tissue HSPGs. In pilot experiments, we have rinsed tissue sections with
2 M NaCl before fixation to remove potential blocking ligands. No difference in binding
patterns is detected, but this possibility cannot entirely be excluded. Despite these limita-
tions, the ligand and soluble receptor binding assays provide important information on
HSPG functions in vivo. These are tools to formulate hypotheses that can subsequently
be vigorously tested with in vitro models.
544 Friedl et al.
Localization of FGF-specific HSPGs 545
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Fig. 2. (opposite page) Binding of FGF-2 and FR1c-AP soluble receptor alkaline phos-
phatase fusion protein to sections of human skin. Tissue HS is identified in frozen sections
(A–F) or paraffin sections (G, H) of human skin. Signal is detected by immunofluorescence
(A–F), or immunoperoxidase (G, H). (A) Detection of all HSPGs with antibody 3G10 after
heparitinase digestion. (B) Binding of biotinylated FGF-7 (200 nM). (C) Binding of FGF-2
(10 nM). (D) Binding of FGF-2, but digestion of tissue section with heparitinase before growth
factor incubation step. (E) Binding of soluble receptor FR1c-AP (30 nM) to tissue section pre-
incubated with FGF-2 (10 nM). (F) Binding of soluble receptor FR1c-AP (30 nM) to tissue
section omitting the FGF-2 incubation step. (G) Binding of FGF-2 (10 nM) to paraffin section
of skin. (H) Binding of soluble receptor FR1c-AP (10 nM) to paraffin section of skin
preincubated with FGF-2 (10 nM). Magnification: 400× (A-F), see also scale bar; 200× (G, H).
Abbreviations: BM, basement membrane; K, keratinocytes; MC, mast cell; V, blood vessels).
(A,C,E) reproduced with permission from Chang, Z., et al. (2000) FASEB J 14, 137–144. (B)
reproduced with permission from Friedl, A., et al. (1997) Am. J. Pathal. 150, 1443–1455.
546 Friedl et al.
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