BRIEF COMMUNICATION

Improved protocol for processing stented porcine coronaryarteries for immunostaining

Arun H. S. Kumar • Scott D. McCauley •

Brian G. Hynes • John O’Dea • Noel M. Caplice

Received: 15 December 2010 / Accepted: 20 January 2011 / Published online: 9 February 2011

� Springer Science+Business Media B.V. 2011

Abstract Percutaneous coronary intervention has resul-

ted in a paradigm shift in the treatment of coronary artery

disease and myocardial infarction. However, neither bare-

metal stents nor polymer-coated drug-eluting stents repre-

sent ideal therapies at this time due to the undesired

in-stent stenosis or delayed thrombosis. Hence there is

pressing clinical need for greater understanding of the

cellular mechanisms involved. It is hoped that this in turn

will provide insight into designing and developing the next

generation of stents. Although immunohistochemistry and

immunofluorescence are appropriate tools in understanding

the molecular histology, performing these techniques on

stented blood vessels is technically challenging because of

poor permeability of antibodies into the stented blood

vessels which are embedded in methacrylate-based resins

and inadequate image resolution due to autofluorescence.

Hence there is a need to develop techniques which can

facilitate immunohistochemistry/immunofluorescence pro-

cedures on stented blood vessel cross-sections. In this study

we describe an improved protocol for processing stented

porcine coronary arteries for immunostaining with smooth

muscle cell, endothelial cell, monocyte and macrophage

markers. We first identified the optimal conditions for resin

embedding of stented artery and cross sectioned the vessels

using high speed precision wafering diamond blade. The

sections were then ground using two levels of water

sandpaper on a Metaserve 2000 grinder to achieve the

desired thickness. For immunostaining, we developed a

novel deplasticization protocol which favors optimal anti-

body permeabilization. Our protocol not only provides

feasibility of improved immunostaining of stented artery

sections but also results in high quality images.

Keywords Stent � Immunofluorescence � Porcine

coronary artery � In-stent restenosis

Introduction

Percutaneous coronary intervention treatment of myocar-

dial ischemia and acute coronary syndromes is extensively

used in the interventional cardiology clinical practice.

Although, stenting of arterial lesions has improved the

outcomes when compared to traditional balloon angio-

plasty (Hudson et al. 2010) in-stent restenosis (ISR)

remains a significant problem, particularly with bare metal

stents (BMS) (Mitra and Agrawal 2006). This has led to the

development of drug-eluting stents (DES) as an alternative;

however, the increased rate of late-stent thrombosis (LST)

with the use of DES is also a concern (Stahli et al. 2009).

Hence it has become necessary to understand the molecular

mechanisms responsible for these limitations so as to

develop better approaches for cardiac interventions in

future.

Stenting of porcine coronary arteries provides important

preclinical model not only to evaluate novel stents for

efficacy and safety but also facilitates understanding of the

mechanisms involved in ISR and LST. Unfortunately, the

precise cellular mechanisms underlying both ISR and LST

are still incompletely understood. This has been in part due

to technical limitations in histological evaluation of stented

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10735-011-9316-8) contains supplementarymaterial, which is available to authorized users.

A. H. S. Kumar � S. D. McCauley � B. G. Hynes � J. O’Dea �N. M. Caplice (&)

Centre for Research in Vascular Biology (CRVB), Biosciences

Institute, University College Cork, College Road, Cork, Ireland

e-mail: n.caplice@ucc.ie

123

J Mol Hist (2011) 42:187–193

DOI 10.1007/s10735-011-9316-8

artery sections (Rippstein et al. 2006). To accurately

characterize the profile of the cells contributing to the neo-

intima formation leading to ISR, use of specific antibodies

to smooth muscle cell, endothelium, macrophages, mono-

cytes and other cell specific markers is required (Jonas

et al. 2005). However, a prerequisite for a good staining

process is a good tissue section of the stented artery. This

challenge has been over come in the recent years by the

development of methyacrylate resins which provides con-

siderable advantage over traditional paraffin or OCT

embedding techniques when sectioning stented arteries

using saw and grinding (SG) techniques (Brasen et al.

2001; Rippstein et al. 2006). Although this improved

methodology has considerably advanced the quality of

stented artery sections which are suitable for classical

histological staining, the possibility of high quality

immunostaining (immunofluorescence) remains a chal-

lenge. While several investigators have described immu-

nostaining, the images acquired had poor resolution and

were associated with a high degree of autofluorescence

(Malik et al. 1998; Brasen et al. 2001; Jonas et al. 2005;

Rippstein et al. 2006). Achieving good immunostaining of

stented arterial sections will facilitate understanding the

molecular histology and cellular dynamics of ISR. A major

barrier to achieving good immunostaining is the use of high

dense methyacrylate resin which is difficult to completely

deplasticize. Consequently the permeabilization of the

antibody becomes problematic and limits the quality of

immunostaining. To address this limitation we optimized

an ideal combination of commercially available methac-

rylate-based resin and developed an improved deplastici-

zation protocol, which provides significantly improved

quality of immunostaining. In this study we outline a

detailed protocol for the successful processing of stented

porcine coronary arteries to obtain improved immunoflu-

orescence labeling.

Materials and methods

Protocol for stenting porcine coronary artery

Female Landrace pigs (25–30 kg; n = 4) were anaesthe-

tized using single intramuscular injection of a combination

of Xylazine (2 mg/kg), and Ketamine (15 mg/kg). The

anaesthesia was maintained using Isoflurane (1.5–2% iso-

flurane delivered via oxygen as a carrier gas). A 5 cm

incision was made 2 cm lateral and parallel to the trachea

and by blunt dissection the carotid artery was exposed and

secured. An arterial cut down was performed in order to

provide access for interventional sheath placement to

facilitate the insertion of balloon catheter and stent

placement. A Philips fluoroscopy C-arm was used to

visualize the location and guide balloon and stent to its

appropriate position in the left anterior descending coro-

nary artery. The bare metal stent (2.5 9 15 mm) was

deployed in the appropriate position with the stent to artery

ratio of 1.1:1. After the stent placement check angiography

confirmed TIMI-3 flow in the stented artery, following

which the guide and stent delivery equipment were with-

drawn under fluoroscopy. The arteriotomy site was then

closed using 8/0 sutures and the surgical wound was closed

using the 4/0 sutures and smeared with betadine. Eight

weeks post procedure the pigs were euthanized by overdose

of pentobarbital and the coronary artery with stent was

dissected out and fixed in chilled 4% paraformaldehyde for

24 h at 4�C.

Embedding of stented coronary artery in resin

Paraformaldehyde fixed stented coronary artery was

washed for 4 h in chilled PBS containing 6.8% sucrose at

4�C and then dehydrated in 100% acetone for 60 min at

4�C. The stented artery was transferred to a 5 ml poly-

styrene tube consisting of infiltration solution (100 ml base

liquid Technovit 8100 mixed with 0.4 g hardener I;

Heraeus Kulzer GmbH, Wehrheim/Ts, Germany) for

overnight at 4�C and then the stent was embedded in the

resin by mixing 0.15 ml of hardener II (Heraeus Kulzer

GmbH, Wehrheim/Ts, Germany) with 5 ml of infiltration

solution. The polystyrene tube with the stent and resin was

sealed with an air tight cap and left at 4�C overnight for

hardening.

Sectioning grinding of the resin embedded stent

After embedding the stented arteries in the resin, a Buehler

IsoMet 5000 high-speed precision saw (Buehler; Dussel-

dorf, Germany) was used to cut cross sections of the

stented artery. The resin block was removed from the

polystyrene tube by cutting it with a hand saw. The resin

block was cleaned and dried using a filter paper and then

mounted perpendicular on to glass slides using super glue

(Bostik, UK). A tiny drop was used, and the block was held

in place by hand under consistent pressure for a minimum

of 30 s to ensure even contact with the slide, and to avoid

the formation of bubbles underneath. The block was then

secured on to a holder in the saw, and the blade was

adjusted to a cutting width of 1 mm. A wayfaring diamond

blade (12.7 9 0.4 mm; Buehler; Dusseldorf, Germany)

was used at a cutting speed of 1,200 rpm with a blade-feed

rate of 10.1 mm/min. During the sectioning, water was

continually jetted on to the cutting surface as a coolant to

minimize friction heat. This resulted in a 100 lm thick

section on the glass slide. The protocol was repeated to cut

the entire length of the stent.

188 J Mol Hist (2011) 42:187–193

123

The 100 lm thick cross section of the stented artery was

ground using two levels of water sandpaper on a Metaserve

2000 grinder (Buehler, Germany). The sections were ini-

tially ground using P800 grit to achieve the desired thick-

ness, 7–10 lm, which took approximately 20 s at a speed of

60 rpm, placing the sample on the sandpaper intermittently

at 1 s intervals. The slides were then smoothed and polished

using P2500 grit paper at 200 rpm for 10 s, at 1 s intervals.

Deplasticization of the stented cross section

Prior to both histological and immunofluorescence stain-

ing, slides were deplasticized to remove excess embedding

resin. Deplasticization was achieved by placing the slides

in Xylene for 20 min twice, 2-methoxyacetate (2-MEA) for

10 min twice, 70% ethanol for 10 min, distilled water

for 10 min, and finally phosphate buffered saline (PBS) for

10 min. It was vital to continually check on the slides

throughout the steps, especially during the 2-MEA

immersion, to ensure the sample did not lift off the slide. If

the sample appeared to be coming off, it was removed from

the solvent and placed in 70% ethanol, continuing with the

steps as described above. All steps were carried out at room

temperature. Once the slides were in PBS, they were either

stained directly for morphometric analysis using histolog-

ical stains or further prepared for immunofluorescence by

an antigen-retrieval protocol.

Immunofluorescence labeling

Prior to immunofluorescence staining, superior results were

achieved by performing antigen retrieval and permeabi-

lizing the tissues to maximize antibody binding. This was

achieved by placing the samples in 4% paraformaldehyde

(PFA) for 10 min at 4�C. Slides were then washed twice in

PBS for 5 min, then placed in a Coplin jar containing

1 mM EDTA (pH 8), and submerged in a boiling water

bath for 15 min. This was followed by 10 min in chilled

methanol at -20�C.

Deplasticized and permeabilized tissue section bound-

aries were marked with a wax pen. They were then incu-

bated in blocking buffer (10% normal goat serum in 0.2%

Triton-X 100 PBS) for 20 min at room temperature with

shaking. This was followed by incubation with primary

antibodies diluted in blocking buffer overnight at 4�C.

Primary antibodies used were: rabbit anti-SMA (Abcam,

1:100), rabbit anti-Calponin (Abcam, 1:100), rabbit anti-

eNOS (Abcam, 1:50), rabbit anti-CX3CR1 (Prosci, 1:50),

and rat anti-F4/80 (Prosci, 1:50), along with appropriate

IgG controls at 1:50 dilution. Sections were then washed in

PBS for 5 min three times, and exposed to fluorescent-

labeled secondary antibodies (goat anti-rabbit/rat 546/488)

diluted in blocking buffer to 1:250/500 for 40 min at room

temperature in humidified chambers with shaking. After

three 5 min PBS washes, slides were incubated with DAPI

at 1:1,000 in distilled water for 10 min, washed in PBS in a

Coplin jar for 5 min, then dried, and covered with anti-

fade, coverslip and sealed with nail polish. The immuno-

stained sections were imaged using Nikon confocal

microscope (Nikon eC1 plus, TE2000E).

Hematoxylin and Eosin (H&E) staining

For H&E staining, deplasticized slides were placed on a

surface shaker in Mayer’s hematoxylin contained in a

Coplin jar for 10 min, followed by rinsing in running tap

water for 15 min. They were then placed in Eosin stain for

10 s, then washed in 70% ethanol for 5 s and cleared in

Xylene for 5 min. Slides were then allowed to air dry

before mounting of a coverslip with DPX prior to imaging

using Nikon upright microscope (Nikon Eclipse 80i).

Results

Optimization of the resin embedding of stented artery

One of the major limitations in the processing of stented

artery for immunofluorescence staining is the hardness of

the resin. The quality of immunostaining is inversely pro-

portional to the hardness of the resin. However, the hard-

ness can only be compromised to an extent which allows

for its cutting without distorting the embedded stented

artery. Hence we opted for a moderate resin strength which

would provide us just enough hardness to hold the stented

artery intact during the process of cutting. This was

achieved by using a milder infiltration solution (i.e., 100 ml

base liquid Technovit 8100 mixed with 0.4 g hardener I in

comparison to the suggested 0.6 g hardener I by Heraeus

Kulzer GmbH, Wehrheim/Ts, Germany) followed by a

milder resin hardener (i.e., 0.15 ml instead of 0.17 ml of

hardener II mixed with 5 ml of infiltration solution). This

protocol allowed us to consistently achieve adequate

hardness for comfortable sectioning of 100 lm thick resin

embedded stented artery without distorting its structure

(Supplement Fig. 1A). Our approach allowed us to look

into the finer microvascular structures within the plaque

(Supplement Fig. 1A) and as well as mononuclear cell

aggregates and smooth muscle coverage near the stent stud

(Supplement Fig. 1B).

Immunofluorescence labeling of stented artery

cross-sections

The sections ground to 7–10 lm thickness and deplasti-

cized were used for immunostaining. The main cell types

J Mol Hist (2011) 42:187–193 189

123

visualized were endothelial cells, vascular smooth muscle

cells, monocytes and macrophages as these cells are

directly relevant to the pathophysiology of instent reste-

nosis. Our protocol resulted in good immunofluorescence

staining for smooth muscle cell markers i.e., a-smooth

muscle actin (Fig. 1) and Calponin (Supplement Fig. 1C)

without any interference from autofluorescence or non-

specific staining (IgG control panel of Fig. 1). The neoin-

tima as expected was predominantly composed of smooth

muscle cells with patches of non smooth muscle regions.

To further test the suitability of our protocol for immuno-

staining we additionally stained for eNOS (endothelial cell

marker, Fig. 2), CX3CR1 (Monocyte specific marker,

Fig. 3a) and F4/80 (macrophage specific marker, Fig. 3b)

and in all cases we achieved good quality immunostaining

with minimal non-specific staining or autofluorescence

(Figs. 2c, 3d). Interestingly macrophages were found as

aggregates near the stent studs in addition to be

Fig. 1 Immunofluorescence staining of stented porcine coronary

artery. Representative cross section of the porcine coronary artery

stented with bare metal stent (2.5 9 15 mm) for 8 weeks were

stained for smooth muscle actin (SMA; green; top right panel). Nuclei

were stained using DAPI (blue; top left panel). Bottom left panel

shows the merged image of SMA and DAPI staining. Rabbit IgG was

used as control (bottom right panel). Adventitia (A), media (M),

neointima (NI). Magnification 2009

190 J Mol Hist (2011) 42:187–193

123

sporadically diffused in the neointima (Fig. 3b). While

CX3CR1 which is expressed on monocytes and as well as

activated smooth muscle cells had diffused staining pattern

(Fig. 3a).

Discussion

The detailed investigation into the origins of ISR and LST

have been hampered in part by technical limitations in the

processing of stented artery tissue, particularly the prepa-

ration of tissue sections that are amenable to immunoflu-

orescence staining. This study describes a new improved

protocol to evaluate stented coronary arteries using a saw

grinding sectioning method, followed by deplasticization

and antigen-retrieval steps modified from those previously

described (Malik et al. 1998; Rippstein et al. 2006).

The primary drawback with the saw and grinding

method of tissue preparation has been the deplasticizing

step that is essential for immunostaining of the stented

tissue sections. The solvents that are required to remove the

resin frequently interact with the super glue used to secure

the specimen to the slide, causing them to become

detached. We found following the manufacturers instruc-

tion of 3 9 20 min in Xylene, 1 9 20 min in 2-MEA,

followed by 5 min twice in both acetone and distilled water

too aggressive (Ali et al. 2007), resulting in tissue loss.

This study uses a milder protocol with 2 9 20 min in

Xylene followed by 2 9 10 min in 2-MEA with continual

observation of the section during the 2-MEA immersion,

and immediate removal and immersion in 70% ethanol if

the tissue begins to detach. Although this resulted in a

variable amount of resin being removed between samples,

it provided the best imaging results after immunostaining.

Another unique element to our protocol is the antigen

retrieval/tissue permeabilization step. We employed a

technique of further fixing tissue sections with 4% para-

formaldehyde and subjecting them to treatment with 1 mM

EDTA in a boiling water bath, and chilled methanol at

-20�C, for 15 and 10 min respectively. This was opti-

mized first on carotid sections and then applied success-

fully to the stented tissue. Previous studies have employed

a similar protocol (Brasen et al. 2001) but none have ever

been as extensive, and we feel this is a critical aspect of the

processing which has allowed for improved immunofluo-

rescence images.

Fig. 2 Immunostaining for endothelial nitric oxide synthase (eNOS)

on stented porcine coronary artery cross sections. Endothelial cells

were labeled with rabbit anti-eNOS (green) a and b panel show eNOS

staining pattern near stent stud and luminal end of the coronary artery,

respectively. c Rabbit IgG was used as control. Nuclei were stained

using DAPI (blue). Magnification 4009

b

J Mol Hist (2011) 42:187–193 191

123

The IF imaging of histological markers for smooth

muscle, endothelial, and macrophage cells is also unique to

this study. These are the major cell types involved in the

ISR and stent re-endothelization process (Ali et al. 2007;

Joner et al. 2008; Stahli et al. 2009). Previous studies have

shown successful immunofluorescence staining of smooth

muscle actin (SMA) and proliferating cell nuclear antigen

(PCNA) in stented human necropsy samples (Rippstein

et al. 2006), and SMA, vascular endothelial growth factor

(VEGF), and flt-1 in stented porcine coronary arteries

(Shibata et al. 2001), though stent struts were removed in

both cases. The images obtained in these studies were

variable in quality, thus making detailed analysis difficult.

We feel our protocol has expanded findings in these studies

Fig. 3 Immunostaining for monocytes and macrophages on stented

porcine coronary artery cross sections. a Myeloid cells were stained

using rabbit anti-CX3CR1 antibody (Green) while macrophages

(b) were stained with rat anti-F4/80 antibody (red; arrows). Panel c is

the merged image of a and b. d Rabbit (Rbt) and Rat IgG was used as

control. Nuclei were stained using DAPI (blue). Magnification 6009

192 J Mol Hist (2011) 42:187–193

123

to provide an improved method of processing stented artery

for immunofluorescence imaging.

Considering the clinical burden of both ISR and LST, it

is critical that strategies be developed to tackle both events,

something which requires a detailed knowledge of the

vascular biology which immunofluorescence imaging can

help provide. Our modified stented artery processing pro-

tocol provides an enhanced method of performing immu-

nofluorescence staining on stented vascular tissue with the

stent strut and tissue structure unaltered. Future studies

focused on specific cellular processes of ISR and LST may

exploit this methodology.

Acknowledgments This work was supported by grants from Sci-

ence Foundation Ireland, Dublin, Ireland (R11482 and RFP06-NMC),

Irish Heart Foundation, Dublin, Ireland (R12348-BH and AHSK), and

HRB summer research fellowship (SM). There are no conflicts of

interests to report.

References

Ali ZA, Alp NJ, Lupton H, Arnold N, Bannister T, Hu Y, Mussa S,

Wheatcroft M, Greaves DR, Gunn J, Channon KM (2007)

Increased in-stent stenosis in ApoE knockout mice: insights from

a novel mouse model of balloon angioplasty and stenting.

Arterioscler Thromb Vasc Biol 27:833–840

Brasen JH, Kivela A, Roser K, Rissanen TT, Niemi M, Luft FC,

Donath K, Yla-Herttuala S (2001) Angiogenesis, vascular

endothelial growth factor and platelet-derived growth factor-

BB expression, iron deposition, and oxidation-specific epitopes

in stented human coronary arteries. Arterioscler Thromb Vasc

Biol 21:1720–1726

Hudson PA, Kim MS, Carroll JD (2010) Coronary ischemia and

percutaneous intervention. Cardiovasc Pathol 19:12–21

Jonas M, Edelman ER, Groothuis A, Baker AB, Seifert P, Rogers C

(2005) Vascular neointimal formation and signaling pathway

activation in response to stent injury in insulin-resistant and

diabetic animals. Circ Res 97:25–33

Joner M, Nakazawa G, Finn AV, Quee SC, Coleman L, Acampado E,

Wilson PS, Skorija K, Cheng Q, Xu X, Gold HK, Kolodgie FD,

Virmani R (2008) Endothelial cell recovery between comparator

polymer-based drug-eluting stents. J Am Coll Cardiol

52:333–342

Malik N, Gunn J, Holt CM, Shepherd L, Francis SE, Newman CM,

Crossman DC, Cumberland DC (1998) Intravascular stents: a

new technique for tissue processing for histology, immunohis-

tochemistry, and transmission electron microscopy. Heart

80:509–516

Mitra AK, Agrawal DK (2006) In stent restenosis: bane of the stent

era. J Clin Pathol 59:232–239

Rippstein P, Black MK, Boivin M, Veinot JP, Ma X, Chen YX,

Human P, Zilla P, O’Brien ER (2006) Comparison of processing

and sectioning methodologies for arteries containing metallic

stents. J Histochem Cytochem 54:673–681

Shibata M, Suzuki H, Nakatani M, Koba S, Geshi E, Katagiri T,

Takeyama Y (2001) The involvement of vascular endothelial

growth factor and flt-1 in the process of neointimal proliferation

in pig coronary arteries following stent implantation. Histochem

Cell Biol 116:471–481

Stahli BE, Camici GG, Tanner FC (2009) Drug-eluting stent

thrombosis. Ther Adv Cardiovasc Dis 3:45–52

J Mol Hist (2011) 42:187–193 193

123