LABORATORY SCIENCES Expression of Vascular Endothelial ... · ide solution for 15 minutes to eliminate endogenous peroxide activity and washed in Tris-buffered saline (pH 7.6) for

LABORATORY SCIENCES

Expression of Vascular Endothelial Growth Factorin RetinoblastomaCarolina Arean, MD; Maria E. Orellana, MD; Daniel Abourbih, BSc; Carmen Abreu, BA;Imelda Pifano, MD; Miguel N. Burnier Jr, MD, PhD

Objectives: To investigate the immunohistochemicalexpression of vascular endothelial growth factor(VEGF) and to determine its possible association withtumor differentiation status, optic nerve and/orchoroidal invasion, anterior chamber invasion, vitre-ous seeding, and basophilic staining of the vascularwalls.

Methods: A retrospective study was performed toidentify the expression of VEGF in 47 of 129 consecu-tive patients with retinoblastoma treated at the OcularPathology Laboratory of the Anatomy and PathologyInstitute of the Central University of Venezuela inCaracas from January 1, 2000, through December 31,2007.

Results: A positive correlation between VEGF staining in-tensity and time of progression and mitotic and apoptoticindexes was observed. However, no correlation was foundbetween VEGF expression and other prognostic factors inthis malignant neoplasm, including tumor stage as as-sessed by the Grabowski and Abramson classification.

Conclusions: Although the isolated characterization ofVEGF in retinoblastoma is not grounds for this proteinto be considered a prognostic factor, its association withmitotic and apoptotic indexes suggests it may play a rolein the progression of this disease. Thus, therapeutic tar-geting of VEGF in retinoblastoma may be an effective strat-egy to reduce tumor progression.

Arch Ophthalmol. 2010;128(2):223-229

R ETINOBLASTOMA IS THE

most common intraoculartumor in children: it rep-resents 3% of the malig-nan t neop l a sms tha t

occur in patients younger than 15years1,2 and less than 1% in all pediatricgroups.3 Retinoblastoma is a rare tumorwith an incidence that varies from 1 per15 000 to 1 per 34 000 live births.1 Inthe United States, retinoblastoma has anincidence of 11 new cases per millionchildren younger than 5 years.4 Retino-blastoma is a congenital neoplasmderived from primitive neuroectodermalcells with retinal differentiation.5 Inpoorly developed countries, this malig-nant neoplasm is typically diagnosed atadvanced stages, between 16 monthsand 2 years of age.4

The retinoblastoma gene is a tumorsuppressor located on the long arm ofchromosome 13. Retinoblastoma devel-opment requires loss or inactivation ofboth alleles.6,7 Retinoblastoma has dis-tinctive histopathologic characteristics:

presence of sleeves surrounded byviable cells and necrotic tissue peripher-ally8; small, round malignant cells withretinal differentiation, in some casesfleurettes; and Flexner-Wintersteinerand Homer-Wright rosettes. The termHomer-Wright rosettes denotes less-differentiated tumors.5 Poor prognosisof this malignant neoplasm is associatedwith invasion of the optic nerve andmeninges and choroidal involvement.5

The importance of angiogenesis intumor growth has been known fordecades, and recently several keyadvances have been made in the devel-opment of antiangiogenic therapies.9

Nevertheless, few studies have investi-gated the relationship between tumorangiogenesis and the prognosis of reti-noblastoma. The purposes of this retro-spective study were to investigate theimmunohistochemical expression ofvascular endothelial growth factor(VEGF) and to determine its possibleassociat ion with known cl inical-histopathologic characteristics.

Author Affiliations: OcularPathology Section, Dr. Jose A.O’Daly Anatomy and PathologyInstitute, Central University ofVenezuela, Caracas(Drs Orellana, Arean, andPifano and Ms Abreu); andHenry C. Witelson OcularPathology Laboratory, McGillUniversity, Montreal, Quebec,Canada (Drs Orellana andBurnier and Mr Abourbih).

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METHODS

PATIENTS

A large series of patients treated at the Ocular Pathology Labo-ratory of the Anatomy and Pathology Institute of the CentralUniversity of Venezuela in Caracas was reviewed, from which47 patients were selected for further immunohistochemicalanalysis. This series represented patients with disease from eachof the 6 stages (Table 1) based on the Grabowski and Abram-son classification.11 Histopathologic reports were reviewed toobtain the following data: age, sex, time between initial symp-toms and enucleation, and pathologic staging.

TISSUE SAMPLES

All samples were previously fixed in 10% buffered formalin, de-hydrated with alcohol solutions and xylene, and paraffin embed-

ded. The cut sections were 4 µm thick and were stained withhematoxylin-eosin,periodicacid–Schiff, andGomori’s trichromestain(originalmagnifications,�100,�100,and�120,respectively).

MICROSCOPIC CLASSIFICATION

Two independent pathologists (M.E.O. and C. Areán) re-viewed the slides by light microscopy and reported the pat-tern of tumor growth (endophytic, exophytic, mixed, ne-crotic, or spontaneous regression), cell differentiation status(well differentiated: foci of Flexner-Wintersteiner rosettes orfleurettes; poorly differentiated: absence of these structures; mod-erately differentiated: isolated Homer-Wright rosettes), opticnerve and choroidal invasion (according to the Khelfaouiprotocol),10 anterior chamber invasion, presence of vitreous seed-ing, and vascular basophilic staining.

IMMUNOHISTOCHEMICAL STUDY

Immunostaining was performed on specimens from the 47 se-lected patients according to conventional protocols, using thestreptavidin-biotin method. Sections (4 µm) of formalin-fixed, paraffin-embedded tissues mounted on poly-L-lysine–coated slides were deparaffinized in xylene and rehydratedthrough serial baths of alcohol and water. The slides were thenplaced in a target retrieval solution with high pH (pH 9.9), andantigen retrieval was performed overnight at 40°C. The hy-drated sections were then treated with a 3% hydrogen perox-

Table 1. Clinicohistopathologic Characteristicsin 129 Patients With Retinoblastomaa

Characteristic No. (%)

Age, mean (SD), mo 29.0 (12.0)Sex

Male 69 (53.4)Female 56 (43.4)Not available 4 (3.1)

Affected eyeRight 60 (46.5)Left 57 (44.2)Bilateral 10 (7.8)Not available 2 (1.6)

Interval onset of symptoms and enucleation, mo0-6 36 (27.9)7-12 24 (18.6)13-23 32 (24.8)�24 10 (7.8)Not available 27 (20.9)

DifferentiationWell differentiated 16 (12.4)Moderately differentiated 24 (18.6)Poorly differentiated 81 (62.8)Necrotic 8 (6.2)

Growth patternMixed 110 (85.3)Endophytic 13 (10.1)Exophytic 2 (1.6)Regression 3 (2.3)Diffuse 1 (0.8)

Anterior chamber invasionPositive 32 (24.8)Negative 97 (75.2)

Choroidal invasionb

I 26 (20.2)II 14 (10.9)III 63 (48.8)IV 15 (11.6)V 11 (8.5)

Optic nerve invasionb

I 36 (27.9)II 39 (30.2)III 30 (23.3)IV 22 (17.1)Not available 2 (1.6)

(continued)

Table 1. Clinicohistopathologic Characteristicsin 129 Patients With Retinoblastomaa (continued)

Characteristic No. (%)

Vitreous seedingPositive 109 (84.5)Negative 20 (15.5)

Basophilic stainPositive 32 (24.8)Negative 97 (75.2)

Mitotic index, per 10 HPF0-5 13 (10.1)6-10 26 (20.2)11-20 49 (38.0)�21 28 (21.7)Not available 13 (10.1)

Apoptotic index, per 10 HPF0-20 15 (11.6)21-40 25 (19.4)41-60 39 (30.2)61-80 20 (15.5)81-100 13 (10.1)�101 4 (3.1)Not available 13 (10.1)

Stagec,d

Ia 13 (10.1)Ib 8 (6.2)Ic 56 (43.4)IIb1 27 (20.9)IIb2 19 (14.7)IIIa 6 (4.7)Not available 0

Abbreviation: HPF, high-power fields.aPercentages may not total 100% owing to rounding. Data are presented

as number (percentage) of patients unless otherwise indicated.bAccording to the protocol of Khelfaoui.10

cAccording to Grabowski and Abramson.11

dNumber (percentage) refers to number of eyes.



ide solution for 15 minutes to eliminate endogenous peroxideactivity and washed in Tris-buffered saline (pH 7.6) for 5 min-utes. The primary antibody used in this study, for 1 hour, wasa polyclonal antibody to VEGF (A-20; Santa Cruz Biotechnol-ogy Inc, Santa Cruz, California).

The monoclonal antibody–treated slides were then rinsed in aphosphate-buffered saline solution for 5 minutes and incubatedwith secondary antibody (Biotinylated Link Universal, DakoCy-tomationLSAB�System-HRP;DakoNorthAmerica Inc,Carpin-teria,California) for20minutesandstreptavidin–horseradishper-oxidase for 20 minutes. Intermittent washing between steps wasperformed with Tris-buffered saline (pH 7.6), and the color wasdevelopedwithfreshlypreparedsolutionof3,3�-diaminobenzidinetetrahydrochloridefor10minutes,posteriorlycontrastedwithMeyerhematoxylin-eosin(1dip).Healthyrenal tissuewasusedasacon-trol,withvesselsthatshowedstrongVEGFimmunoreactivity.Nega-tive controls included the omission of the primary antibody andsubstitution with nonimmune serum.

IMMUNOSTAINING ANALYSIS ANDSTATISTICAL ANALYSIS

For immunostaining analysis, tumors were scored by assess-ment of the proportion and intensity of stained tumor cells andthen scored by a semiquantitative method. The presence of cy-

toplasmic staining was considered a positive finding. Posi-tively stained cells were counted in 10 randomly selected fieldsunder a magnification of �40. Intensity was graded as nega-tive, weak, moderate, or intense, and the percentage of stainedcells was classified as less than or equal to 50.0%, 51.0% through75.0%, or more than 75.0%.

For statistical analysis of the results, we calculated fre-quency and percentage for nominal and ordinal variables. Datawere analyzed for statistical significance using the Pearson �2

test and the Pearson product-moment correlation coefficient(P� .05 was considered significant).

RESULTS

Clinicohistopathologic data for the 129 patients in thestudy are given in Table 1. There were more male thanfemale patients (1:0.82). The most common symptomsrecorded were leukocoria, strabismus, diminished vi-sual acuity, and orbital mass.

A mixed-type pattern of growth was seen in 110 pa-tients, and the endophytic pattern was seen in 13 pa-tients. Other patterns (regression, exophytic, and dif-fuse) were seen infrequently (Figure 1A and B).

A B

C D

Figure 1. Retinoblastoma. A, Mixed endophytic-exophytic growth pattern. B, Exophytic growth pattern. C, Well-differentiated tumor with Flexner-Wintersteinerrosettes (hematoxylin-eosin, original magnification �200). D, Poorly differentiated retinoblastoma (hematoxylin-eosin, original magnification �200).



According to the histopathologic results, poorly differ-entiated tumors were the most frequent type (81 pa-tients), followed by moderately differentiated (24 pa-tients) and well differentiated (16 patients) (Figure 1Cand D). Anterior chamber invasion was observed in 24.8%of the patients, whereas 48.8% of the patients had mas-sive choroidal invasion. Compromise of the sclera was

seen in 11.5% of intrascleral cells (stage IV) and 8.6% ofextrascleral cells (stage V), respectively. Assessment ofoptic nerve invasion yielded no remarkable differencesamong the 4 groups. Most patients (84.5%) showed vit-reous seeding (Figure 2).

The mitotic index was variable among the patients;38.0% of patients had 11 to 20 mitoses per 10 high-power fields (HPF), 22.0% had more than 20 mitotic fig-ures per 10 HPF, and 20.0% had 6 to 10 mitoses in 10HPF. Evaluating the apoptotic index, 30.0% of patientshad 41 to 60 apoptotic bodies per 10 HPF, and 19.0%had an apoptotic index of 21 to 40 per 10 HPF.

The immunoreactivity of VEGF was positive in 43 of44 patients (Table 2 and Figure 3). Three of the 47 pa-tients studied could not be evaluated because of extensivenecrosis. No statistically significant correlation was foundbetween VEGF immunostaining and many established reti-noblastoma prognostic factors, such as optic nerve or cho-roidal invasion, anterior chamber invasion, vitreous seed-ing,orbasophilic stainingon thevascularwalls. Inour series,the lack of follow-up prevents the possibility of establish-

A B

C D

Figure 2. Retinoblastoma. A, Vessel surrounded by viable neoplastic cells that form a perivascular sleeve (hematoxylin-eosin, original magnification �200).B, Tumor extrascleral invasion (Gomori trichrome, original magnification �120). C, Postlaminar invasion (Gomori trichrome, original magnification �100).D, Massive invasion of the choroid (hematoxylin-eosin, original magnification �320).

Table 2. Vascular Endothelial Growth Factor Stainingin 44 Patients With Retinoblastoma

Staining No. (%) of Patients

IntensityNegative 1 (2)Weak 7 (16)Moderate 13 (30)Intense 23 (52)

Extent, %�50 5 (12)51-75 14 (33)76-100 24 (56)



ing a correlation between VEGF immunostaining and prog-nosis, even though this variable was not related to the stageof the disease, according to the Grabowski and Abramsonclassification.11

Using the coefficient correlation test, we found a posi-tive correlation between VEGF immunostaining inten-sity and the number of apoptotic bodies (P=.03); thehigher the apoptotic index, the stronger the intensity ofVEGF staining (Figure 4). A relation was found be-tween mitotic index and immunostaining intensity ac-cording to the �2 test (P=.003) (Figure 4). In addition, arelation was found between the intensity of VEGF stain-ing and an independent variable: time with symptomsbefore the enucleation, also known as the interval onsetof symptoms and enucleation (P=.03) (Figure 5).

COMMENT

Retinoblastoma is the most common intraocular tumorin childhood, with an annual mean incidence of 1 in18 000 live births and a mortality rate that varies widelyacross the globe.12,13 In well-developed countries, reti-noblastoma is rarely a life-threatening condition be-cause of early diagnosis, but in underdeveloped and de-veloping countries, clinical diagnosis is made in advancedstages, and the mortality rate remains high.4,13-16

In our study, patients had a mean age at diagnosis of29 months. This value is similar to those from other LatinAmerican and Asian series (32, 31, and 26 months in Ven-ezuela, Mexico, and Taiwan, respectively).4,16,17 The in-terval between the first symptoms and enucleation rangedfrom 15 days to 44 months, with a slightly higher num-ber of patients in the 0-month to 6-month group. Mostof our patients had poorly differentiated tumors, similarto the work of Biswas et al.18

Marback et al15 measured the relative vascular area ofthe tumor in retinoblastomas and compared their re-sults with optic nerve and choroidal invasion. They in-dicated that the relative vascular area of the tumor seemsto be a promising prognostic marker for metastatic dis-ease and should be evaluated in a large sample of eyeswith retinoblastoma.

Previous studies19 have reported that angiogenic po-tential in retinoblastoma correlates with invasive growthand metastasis and that these 2 factors are associated withpoor prognosis. These tumors, however, appear to de-pend on a heterogeneous vasculature composed of an-giogenic neovessels and pericyte-committed mature vas-culature, the latter composed of endothelial cellsdependent on angiogenic factors such as VEGF.

It is known that VEGF messenger RNA is expressedin retinoblastoma neoplastic cells, but little of such ex-

A B

C D

Figure 3. Vascular endothelial growth factor (VEGF) immunostaining of retinoblastoma. A, VEGF negative (original magnification �100). B, Weak positivity(original magnification �200). C, Moderate positivity (original magnification �240). D, Strong positivity (original magnification �320).



pression occurs in tumor endothelial cells, and that VEGF,which is secreted from neoplastic cells, influences nearbyendothelial cells and functions as a paracrine media-tor.20 In vitro studies have shown that VEGF stimulatesendothelial cell division and migration, and its produc-tion is induced by hypoxia.21 This factor has been impli-cated in cellular proliferation of gastrointestinal carci-nomas and in other types of cancer.22

In our series, 98% of the patients tested positive forretinoblastoma by VEGF immunostaining, of which mosthad diffuse and strong staining. Of the 7 patients withwell-differentiated retinoblastoma, 2 showed focal andweak VEGF staining, whereas the other 5 had strong anddiffuse staining. These results differ from those of Keri-mogglu et al,23 who measured angiogenesis (microves-sel density) and found higher levels in poorly differen-tiated retinoblastomas.

We found a statistically significant relation betweenthe interval from the onset of the symptoms to theenucleation and VEGF immunostaining intensity. Thisfinding indicates that the intensity of the immuno-staining depends on the time between the onset ofsymptoms and enucleation. This could suggest thattumors with delayed diagnosis and treatment could

have more angiogenic potential and may be moreprone to dissemination.

High mitotic and apoptotic indexes reflect high pro-liferative activity in any tumor. Kerimogglu et al23 havesuggested that the apoptotic index could be an impor-tant metastatic predictor for retinoblastoma. Most of ourpatients had high mitotic and apoptotic indexes, and wefound a statistically significant correlation between VEGFimmunostaining intensity and the apoptotic index(Figure 4). Moreover, a relation between VEGF immuno-staining intensity and percentage of staining with the mi-totic index was also found. With respect to the apop-totic index, it has been demonstrated that deletion of theretinoblastoma gene produces apoptosis rather than tu-mor formation because the loss of the retinoblastoma genetriggers a p53-mediated apoptotic response.24

No correlation was found between VEGF immuno-staining and tumor staging. This lack of correlation im-plies that all retinoblastomas, not just those limited toretina and cases with choroidal or optic nerve invasion,have the capacity to produce angiogenic factors and byextension induce angiogenesis. Marback et al15 have sug-gested that retinoblastomas with numerous blood ves-sels (measured with the relative vascular area of the tu-mor) are those at an advanced stage of disease. Althoughthe isolated characterization of VEGF in retinoblastomashould not be taken as a prognostic factor, its associa-tion with the apoptotic index suggests a role for this pro-tein in the progression of this disease.

Our data suggest that the use of anti-VEGF therapiesin retinoblastoma may be efficacious in targeting imma-ture neovessels within the tumor. This approach, coupled

4.0

3.0

3.5

2.5

2.0

1.5n = 44r = 0.33P = .03

χ2 = 29.409P = .003

1.0

0 20 40 60 80 120100 140No. of Apoptotic Bodies per 10 HPF

VEGF

Sta

inin

g In

tens

ity

4.0

3.0

3.5

2.5

2.0

1.5

1.0

0 10 20 30 40 50No. of Mitotic Figures per 10 HPF

VEGF

Sta

inin

g In

tens

ity

B

A

Figure 4. Intensity of vascular endothelial growth factor (VEGF) staining.A, Number of apoptotic bodies (�10 high-power fields [HPF]). B, Number ofmitotic figures (�10 HPF).

4.0

2.5

3.0

3.5

2.0

1.5

1.0

0 5 10 15 2520 30Time, mo

Inte

nsity

Figure 5. Time with symptoms before the enucleation and intensity ofvascular endothelial growth factor staining.



with a strategy to treat pericyte-committed mature tu-mor vasculature, may be effective in the management ofthis disease.

Submitted for Publication: February 3, 2009; final re-vision received September 22, 2009; accepted October5, 2009.Correspondence: Maria E. Orellana, MD, Henry C. Wi-telson Ocular Pathology Laboratory, McGill University,3775 University St, Room 216, Montreal, QC H3A 2B4,Canada ([email protected]).Financial Disclosure: None reported.Additional Contributions: Douglas Angulo, MSc, pro-vided valuable statistical analysis.

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2. Hurwitz RL, Shields CLSJ, Chevez-Barrios P, Chintagumpala MM. Retinoblas-toma. In: Pizzo PA, Poplack DG, eds. Principles & Practice of Pediatric Oncol-ogy. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2005:865-886.

3. Abramson DH. Retinoblastoma in the 20th century: past success and future chal-lenges: the Weisenfeld lecture. Invest Ophthalmol Vis Sci. 2005;46(8):2683-2691.

4. Leal-Leal C, Flores-Rojo M, Medina-Sanson A, et al. A multicentre report fromthe Mexican Retinoblastoma Group. Br J Ophthalmol. 2004;88(8):1074-1077.

5. Font RL, Croxatto JO. Tumors of the Retina. Bethesda, MD: American Registryof Pathology; 2007.

6. Melamud A, Palekar R, Singh A. Retinoblastoma. Am Fam Physician. 2006;73(6):1039-1044.

7. Harbour JW. Eye cancer: unique insights into oncogenesis: the Cogan Lecture.Invest Ophthalmol Vis Sci. 2006;47(5):1736-1745.

8. Burnier MN, McLean IW, Zimmerman LE, Rosenberg SH. Retinoblastoma: therelationship of proliferating cells to blood vessels. Invest Ophthalmol Vis Sci.1990;31(10):2037-2040.

9. Rosenblatt MI, Azar DT. Anti-angiogenic therapy: prospects for treatment of ocu-lar tumors. Semin Ophthalmol. 2006;21(3):151-160.

10. Khelfaoui F, Validire P, Auperin A, et al. Histopathologic risk factors in retino-blastoma: a retrospective study of 172 patients treated in a single institution. Cancer.1996;77(6):1206-1213.

11. Grabowski EF, Abramson DH. Intraocular and extraocular retinoblastoma. He-matol Oncol Clin North Am. 1987;1(4):721-735.

12. de Souza Filho JP, Martins MC, Torres VL, et al. Histopathologic findings in reti-noblastoma [in Portuguese]. Arq Bras Oftalmol. 2005;68(3):327-331.

13. Sang DN, Albert DM. Retinoblastoma: clinical and histopathologic features. HumPathol. 1982;13(2):133-147.

14. Shields CL, Shields JA, Baez KA, Cater J, De Potter PV. Choroidal invasion ofretinoblastoma: metastatic potential and clinical risk factors. Br J Ophthalmol.1993;77(9):544-548.

15. Marback EF, Arias VE, Paranhos A Jr, Soares FA, Murphree AL, Erwenne CM.Tumour angiogenesis as a prognostic factor for disease dissemination inretinoblastoma. Br J Ophthalmol. 2003;87(10):1224-1228.

16. Chang CY, Chiou TJ, Hwang B, Bai LY, Hsu WM, Hsieh YL. Retinoblastoma inTaiwan: survival rate and prognostic factors. Jpn J Ophthalmol. 2006;50(3):242-249.

17. Benguigui ABM, Garabito Z, Ramirez G, Arevalo I, Suarez A. Retinoblastoma: re-port of ophthalmic oncology consultation at Francisco Antonio Rısquez Hospitalbetween April 1992 and September 1995 [in Spanish]. Rev Oftalmol Venez. 1998;54(3):43-50.

18. Biswas J, Das D, Krishnakumar S, Shanmugam MP. Histopathologic analysis of232 eyes with retinoblastoma conducted in an Indian tertiary-care ophthalmiccenter. J Pediatr Ophthalmol Strabismus. 2003;40(5):265-267.

19. Jockovich ME, Pina Y, Alegret A, Cebulla C, Feuer W, Murray TG. Heteroge-neous tumor vasculature in retinoblastoma: implications for vessel targetingtherapy. Retina. 2008;28(3)(suppl):S81-S86.

20. Lee SY, Kim DK, Cho JH, Koh JY, Yoon YH. Inhibitory effect of bevacizumab onthe angiogenesis and growth of retinoblastoma. Arch Ophthalmol. 2008;126(7):953-958.

21. Stitt AW, Simpson DA, Boocock C, Gardiner TA, Murphy GM, Archer DB. Ex-pression of vascular endothelial growth factor (VEGF) and its receptors is regu-lated in eyes with intra-ocular tumours. J Pathol. 1998;186(3):306-312.

22. Montero E, Abreu C, Tonino P. Relationship between VEGF and p53 expressionand tumor cell proliferation in human gastrointestinal carcinomas. J Cancer ResClin Oncol. 2008;134(2):193-201.

23. Kerimogglu H, Kiratli H, Dinctürk AA, Söylemezoglu F, Bilgic S. Quantitative analy-sis of proliferation, apoptosis, and angiogenesis in retinoblastoma and their as-sociation with the clinicopathologic parameters. Jpn J Ophthalmol. 2003;47(6):565-571.

24. Murphree MS, Harbour JW. Tumors of the retina. In: Ryan SJ, ed. Retina. Phila-delphia, PA: Elsevier/Mosby Publishing; 2006:557-607.

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LABORATORY SCIENCES Expression of Vascular Endothelial ... · ide solution for 15 minutes to eliminate endogenous peroxide activity and washed in Tris-buffered saline (pH 7.6) for