Wheat germ agglutinin as a counterstain for immunofluorescencestudies of equine hoof lamellae

Robert K. Clark1,2 and Hannah L. Galantino-Homer1

1Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, PA, USA;2STEM and Health Division, Cumberland County College, Vineland, NJ, USA

Correspondence: Hannah Galantino-Homer, Department of Clinical Studies, New Bolton Center, 382 West Street Road, Kennett Square, PA

19348-1692, USA, Tel.: 1-610-925-6246, Fax: 1-610-925-6821, e-mail: hghomer@vet.upenn.edu

Abstract: Equine laminitis is a common, painful, debilitating

condition of the hoof that is a leading cause of disability in

horses, often necessitating euthanasia. The equine hoof represents

an extreme evolutionary adaptation of an epidermal structure

homologous to the human or murine nail units.

Immunohistochemistry is frequently utilized in the study of the

pathophysiology of laminitis. The complex, multilayered,

extensively interdigitated epidermal–dermal lamellar interface

renders precise interpretation of immunofluorescence localization

difficult, especially when effective technique and reagents render

non-reactive tissues completely dark. Fluorescent-conjugated

wheat germ agglutinin (WGA) selectively labels dermal

extracellular matrix fibres and epidermal cell membranes in tissue

sections of horse hoof lamellae, is compatible with indirect

immunofluorescence and augments interpretation of indirect

immunofluorescence antigen localization. The current report

details the use of WGA as a rapid, simple, economical

counterstain for immunofluorescence studies of the equine hoof

and may have application to other complex epidermal tissue

structures.

Key words: counterstain – epidermis – histology – laminitis – lectins

Accepted for publication 6 July 2014

BackgroundEvolutionary adaptation of the horse (Equus caballus) resulted in

elongation of the distal limb, loss of digits and the development of the

hoof, an epidermal adnexal structure homologous to the human or

murine nail units, in which the horse’s weight is transferred from the

distal phalangeal bone to the hoof capsule and hence to the ground

[for review, see (1–3)]. While this arrangement provides numerous

functional advantages including traction and impact absorption, it

requires an exceptional degree of adhesion between epithelial and

mesenchymal tissues. This is accomplished, in part, through the mas-

sive surface area (0.8 m2) afforded by the interdigitating tissues in the

550–600 primary lamellae, each containing 150–200 secondary lamel-

lae, per hoof (1). This complex suspensory apparatus is prone to

failure through an array of metabolic and traumatic pathways leading

to laminitis, a painful, debilitating condition which is the second most

common underlying cause for euthanasia in horses (4).

Indirect immunofluorescence is frequently utilized in the study

of laminitis. One weakness of this technique is the relative inability

to discern the precise location of immunoreactive structures within

the surrounding non-immunoreactive tissues when little or no

background fluorescence renders those components dark. This

could be greatly mitigated through the use of an effective counter-

stain (5,6). During a recent study investigating plant lectin binding

to lamellar tissue, we noted that wheat germ agglutinin lectin

(WGA) demonstrated affinity for extracellular matrix fibres in the

hoof dermis and cell membranes in the epidermis. We observed that

this binding pattern renders WGA potentially useful as a counter-

stain to immunofluorescent studies on the hoof.

Experimental designIn order to make use of WGA as a counterstain to indirect immu-

nofluorescence, it was necessary to ascertain whether this binding

pattern would remain consistent in tissues from a wide range of

horses and when used in combination with primary and secondary

antibodies. See Data S1 for detailed materials and methods. Tis-

sues containing lamellae were obtained and paraformaldehyde

(PFA)-fixed, sucrose-dehydrated, snap-frozen and cryostat-sec-

tioned according to standard protocols. Tetramethylrhodamine

isothiocyanate (RITC)-conjugated WGA (Vector Laboratories,

Burlingame CA, USA) diluted 1:300 in phosphate-buffered saline

(PBS) was used to stain tissue sections.

For WGA/indirect immunofluorescence double labelling, tissue

sections were immunostained using either rabbit anti-desmoplakin

I and II antiserum (ab71690, Abcam Inc., Cambridge, MA, USA)

diluted 1:400 in PBS containing 3% bovine serum albumin (BSA),

or mouse monoclonal anti-keratin KRT14 (clone LL002, Abcam

Inc.) diluted 1:50, as primary antibodies. Secondary antibodies

consisted of Alexa Fluor 488-conjugated goat anti-rabbit (Abcam

Inc.), or goat anti-mouse IgG (Abcam Inc.) antiserum, diluted

1:250 in PBS containing 3% BSA. Sections were examined and

imaged by confocal microscopy.

ResultsWheat germ agglutinin demonstrated the same binding pattern

whether applied alone (not shown) or in combination with indi-

rect immunofluorescence (Figs 1 and 2); epidermal cells through-

out all layers of the secondary epidermal lamellae and keratinized

axis of the primary epidermal lamellae demonstrated linear mem-

branous/submembranous fluorescence, while the dermis, including

primary and secondary dermal lamellae, demonstrated a fluores-

cence pattern consistent with fibres of the extracellular matrix. In

double-labelling experiments, whether the antibody was directed

against the basal cell cytoskeletal protein KRT14 (Fig. 1) (7,8), or

the desmosomal cytolinker desmoplakin (Fig. 2) (9), WGA

fluorescence enhanced the ability to observe the tissue and cellular

localization of the antibody. The pattern of WGA staining was

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons LtdExperimental Dermatology, 2014, 23, 677–678 677

DOI: 10.1111/exd.12495

www.wileyonlinelibrary.com/journal/EXDMethods Letter to the Editor

uniform throughout this series of samples from 21 horses, repre-

senting different breeds and ages ranging from 5 days to 12 years,

as well as laminitic and non-laminitic hooves.

ConclusionsLectins have been used in the characterization of the complex strati-

fied cytoarchitecture of epidermal tissues and adnexae in many spe-

cies including the dog (10), horse (11,12) and human (13,14).

Epidermal WGA binding has been described in several studies.

While Reano et al. (14) reported that acetone-fixed frozen sections

failed to show WGA binding in human epidermis, Virtanen et al.

(13) demonstrated a binding pattern in PFA-fixed frozen human

skin similar to that which we see in the PFA-fixed frozen equine

hoof. As paraffin embedding also eliminates WGA binding (13), it is

possible that these differences are related to tissue preparation tech-

nique. One minor difference between our study and that of Virtanen

et al. is that we observed some binding in the hard keratinized hoof

wall, while they did not see any binding in the comparable skin

epidermal layer, perhaps due to species and tissue differences.

An electron microscopic study of equine scrotal skin apocrine

glands reports a membranous pattern of WGA binding that may

be similar to that which we see in hoof epidermal cells, although

these locations represent very different patterns of epithelial differ-

entiation (11). Another group examined lectin binding in equine

skin and hoof wall and reported only tightly restricted staining

patterns (12). They did not, however, examine hoof lamellae, nor

did they use frozen sections. WGA preincubation has also been

reported to inhibit binding of pemphigus foliaceus autoantibodies

to desmoglein 1 in human tissues, again showing an epidermal

membranous binding pattern (15).

To our knowledge, WGA has not been used as a counterstain to

indirect immunofluorescence labelling of epidermal tissues in any

species. Its utility for this purpose became apparent to us, perhaps

because of the complex tissue architecture of the hoof lamellae and

due to the difficulty of precisely localizing specific antigens in these

tissues. Once noted, the use of WGA as a counterstain enabled us to

precisely elucidate the location of antibody binding with greater ease

and confidence than we had previously been able to do, providing a

rapid, economical and simple counterstain to immunofluorescence

studies on equine hoof lamellae. WGA counterstain may also prove

useful in dermatological immunofluorescence studies, such as

recently published studies of genetic and acquired human epidermal

diseases (16–22), mouse models of human epidermal disease (21,23)

and complex epidermal tissues in other species (24).

AcknowledgementsThe authors thank Christopher Pollitt (Queensland, Australia) for tissue

samples and Gordon Ruthel (Philadelphia, USA) for confocal microscopy

advice. This work was funded by grants from the Bernice Barbour Founda-

tion, Inc. (0048.42008), Grayson-Jockey Club Research Foundation and the

University of Pennsylvania Fund for Laminitis Research.

Author contributionsRKC and HLG-H contributed equally to the conception, design, conduct,

interpretation, writing and editing of the current work.

Conflict of interestsThe authors have declared no conflicting interests.

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Supporting InformationAdditional supporting data may be found in thesupplementary information of this article:Data S1. Detailed Materials and Methods.

(a) (b) (c)

Figure 1. Immunofluorescence histochemistry for keratin KRT14 combined withwheat germ agglutinin (WGA) counterstaining on equine hoof lamellae. Greenfluorescence demonstrates immunolocalization of KRT14 to the cytoplasm of basalcells of the secondary epidermal lamellae (a, b). Red fluorescence demonstrateslocalization of WGA binding to extracellular matrix fibres of the dermis includingthe secondary dermal lamellae and to cell membranes in secondary and primaryepidermal lamellae (b, c). Note that red WGA fluorescence augments the preciselocalization of the green KRT14-positive cells. Asterisk indicates the central axis ofa primary epidermal lamella for orientation. Bar = 25 lm.

(a) (b) (c)

Figure 2. Immunofluorescence histochemistry for desmoplakin combined withwheat germ agglutinin (WGA) counterstaining on equine hoof lamellae. Greenfluorescence demonstrates punctate linear, peri-membranous immunolocalizationof desmoplakin within epidermal lamellae (a, b). Red fluorescence demonstrateslocalization of WGA binding to extracellular matrix fibres in the primary andsecondary dermal lamellae and to cell membranes in primary and secondaryepidermal lamellae (b, c). Note that red WGA fluorescence augments the preciselocalization of the green desmoplakin immunoreactivity. Asterisk indicates thecentral axis of a primary epidermal lamella for orientation. Bar = 25 lm.

678ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Experimental Dermatology, 2014, 23, 677–678

Methods Letter to the Editor