J Clin Pathol 1988;41:1217-1222

Sensitive avidin biotin based technique for identifyingliver membrane antigens in tissue sectionsJ SIPOS, B M McFARLANE, R WILLIAMS, I G McFARLANE The Liver Unit, King's CollegeHospital, London

SUMMARY An enzyme immunohistochemical technique for the localisation of liver membraneantigens in tissue sections by antisera raised in guinea pigs against the liver preparation known as

"liver specific membrane lipoprotein (LSP)" was developed, based on the alkaline phosphataseavidin biotin complex (ABC AP) system. Of a wide range of fixatives and fixation conditionsinvestigated, a short (five minute) exposure of cryostat sections to Bouin's fluid provided the mostsatisfactory results and-together with procedures to block endogenous biotin and alkalinephosphatase-yielded clear sections with no background staining or other artefacts to interfere withspecific staining patterns. The sensitivity ofthe technique approaches that ofa radioimmunoassay, as

shown by the staining of the sinusoidal domains of hepatocellular plasma membranes by the guineapig anti-LSP antisera at dilutions up to 1/50 000. Apart from its reliability and sensitivity theprocedure offers additional advantages over techniques such as indirect immunofluorescence in thatit provides a permanent preparation with well defined morphological details which can be seen byordinary light microscopy.

Liver specific membrane lipoprotein (LSP) is apreparation from normal liver that contains variousmembrane fragments and other macromolecular livercell constituents.' A proportion of the membranefragments derive from the hepatocellular plasmamembrane and clinical interest in LSP has beenstimulated by the numerous reports of cellular andhumoral immune reactions against different antigensin this preparation in patients with autoimmunechronic active hepatitis and certain other liverdiseases.' Current evidence suggests that the antigensin LSP that are of greatest importance, as targets ofautoreactions are both liver specific and immuno-chemically cross reactive among species'4 and, if theautoreactions are associated with pathogenesis, therelevant antigens must presumably be located onhepatocellular surfaces.The immunochemical heterogeneity ofLSP and the

comparative lability of many of its constituentantigens have presented important technical obstaclesto defining the targets of these autoreactions and inparticular to locating them in the liver by histo-chemical techniques. Lebwohl and Gerber wereunable to obtain any liver specific staining ofunfixed frozen sections of human liver by indirectimmunofluorescence with a rabbit antiserum to

jSccepted for publication 2 June 1988

human LSP and speculated that the relevant antigensmay have been present in amounts that were too smallto be detected by this technique.6 Lee et al suggestedthat the antigens might have been lost duringpreliminary washing of the sections and showed thatprevious fixation with Bouin's fluid permitteddetection of linear polygonal immunofluorescencestaining of hepatocyte plasma membranes in rat liversections by serum samples from patients with variousliver diseases."8

Helin et al used the more sensitive peroxidase linkedavidin biotin complex (ABC) system to investigatereactions against deparaffinised sections of paraffinwax embedded normal human liver with rabbit anti-human LSP antisera.9 They were able to stainhepatocyte plasma membranes with some antisera butthe results were variable and it was difficult to interpretthe findings. The present study was therefore under-taken to define more precisely the conditions requiredto identify the location of the relevant target antigensof anti-LSP antibodies in liver sections by using thehighly sensitive alkaline phosphatase ABC system.

Material and methods

PREPARATION OF RAT TISSUESWistar rats were killed by cervical dislocation and

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tissues were excised into ice cold (4°C) phosphatebuffered saline (PBS), pH 7-4, then immediately snapfrozen in isopentane liquid nitrogen according to themethod of Sheibani and Tubbs.'" Five micron sectionsof composite (liver, adrenal cortex, kidney, pancreas,and lung) tissue blocks were cut in a cryostat at- 20°C, mounted on glass slides, air dried for one hourat room temperature, and then stored at - 20°C.

IMMUNOHISTOCHEMICAL PROCEDURESThe procedures described here are the optimalconditions derived from the investigations detailed inthe Results section. The reagents for the avidin biotincomplex (ABC) system (Vector Laboratories Peter-borough, UK) were: avidin biotin blocking kit numberSP-2001; ABC alkaline phosphatase conjugatedantiguinea pig IgG Vectastain kit number AK-5007;and alkaline phosphatase substrate kit number SK-5 100. Unless otherwise indicated, all preparations anddilutions of these reagents were performed strictlyaccording to the manufacturer's instructions. Thaw-ing of tissue sections and all other procedures werecarried out at room temperature.

Sections were fixed by immersion in Bouin's fluid(75 ml saturated aqueous picric acid, 24 ml formalin,and 5 ml glacial acetic acid) for five minutes. Slideswere then transferred directly to PBS and washedthoroughly for 15 minutes with three changes of thebuffer.

All of the following procedures were carried out inthe dark in a humidified chamber. Endogenous biotinin the fixed sections was inactivated by using theVector avidin biotin blocking kit. Thus sections wereincubated for 20 minutes with the avidin solutionprovided, washed with PBS (pH 7.4) for five minutes,then incubated with the biotin solution for 20 minutes.After a further five minutes wash in PBS sections wereincubated with a 1/50 dilution (in PBS) ofnormal goatserum for 20 minutes to block any sites that might bindgoat proteins non-specifically. Excess normal goatserum was removed by blotting and the sections wereincubated for 30 minutes with the required dilution(1/50 or greater, in PBS) of the guinea pig antirat LSPantisera washed for 10 minutes in PBS, incubated withthe goat antiguinea pig IgG for 30 minutes, washed(for 10 minutes with PBS) and then incubated with theVectastain ABC alkaline phosphate reagent. After afurther 10 minute wash in PBS the sections wereincubated with the Vector Red alkaline phosphatasesubstrate solution to which had been added levamisole(1mM final concentration), washed for five minutes intap water, counterstained with Mayer's haemalum for10 seconds and rinsed in tap water. Finally, thesections were dehydrated by absolute ethanol, clearedby xylene, and mounted in distrene plasticiser xylol.

Antisera were raised in guinea pigs against rat LSP

Sipos, McFarlane, Williams, McFarlane

that had been prepared from 100 000 x gsupernatants of normal rat livers by gel filtration oversepharose 6B in 0-1 M boric acid/sodium boratebuffer, pH 8-5, containing 1 mM disodium edetic acidas previously described." The immunisationprogramme which entailed subcutaneous injections ofLSP with and without Freund's complete adjuvantover five weeks has been previously described."Production of anti-LSP antibodies was monitored byradioimmunoassay and titres of the antisera deter-mined by doubling dilution titration.'2

Results

At the beginning of the study preliminary investi-gations were undertaken to test each component ofthe system for non-specific reactivity, starting withthe alkaline phosphatase substrate and workingbackwards through the procedure. As described by themanufacturers, it was found that when unfixed liversections were incubated with the alkaline phosphatasesubstrate alone a definite positive reaction wasobtained (an intense red staining mainly of bile ductepithelial cells and indicating pronounced endogenousalkaline phosphatase activity) and that this could becompletely abolished by including levamisole (at 1mM) in the substrate solution.When the Vectastain ABC reagent was used without

any antiserum, fine, granular staining ofthe cytoplasmwas seen (fig 1). That this could be abolished by usingthe avidin biotin blocking procedure (fig 2a) indicatedthat it was due to binding of the ABC to endogenousbiotin in the cells. It should be noted that a quick rinseafter performing this procedure is inadequate, and aminimum of five minutes washing after each step isessential.

* 4* S

Fig I Definite granular staining ofthe cytoplasm ofhepatocytes seen when endogenous biotin was not blockedbefore incubation with the ABC reagent.

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Incubation of sections with undiluted guinea pigserum (after blocking endogenous biotin) also led tofine granular staining. To investigate the possibilitythat this might have been due to ionic interactions withbasic proteins (which have the ability to bind ABC) or

to interaction with lectin like molecules in the liver thatmay have affinity for carbohydrate residues in guinea

Fig 2 Staining main/v of sinu.soidazl surface.s of hepateso te.vby guinea pig anti-LSP antiserum. a} Control (normal

guinea pig serum at 1.50 dilution rh s)strong granularstaining ith guinea pig anti-LSP at 1 1000 dlilution:(c) fine granular staining with antiserum at 1 50 000

dilution. Developed wvith Vector Red alk-aline phosphatasesubstrate and counterstained with MaYer S haemalumn.

pig serum glycoproteins, the procedure was carriedout using carbonate buffer, pH 9 4, and (in furtherexperiments) after pretreatment of the sections with0 05 M a-methyl-D-mannoside. Neither of thesemodifications had any effect, but it was found that a

completely clear section with no residual staining wasobtained ifthe normal guinea pig serum was applied at

Table 1 Details of the.fixatives andfixation conditions investigated

Fixative Buffer Temperature Time (minutes)

Acetone None - 200C I and 5Acetone None +40C 10Ethanol None - 200C I and 5Ethanol None +4°C 10Methanol None +4°C 10Paraformaldehyde 2 0% Aqueous, pH 7-5 Room temperature 30Formalin 3-7% Phosphate, pH 7-4 Room temperature 0-5, 2, 10, 30Formalin 3-7% Tris, pH 7-6 Room temperature 15Formalin 3-7% with 0-1% Triton X-100 Phosphate, pH 7-4 Room temperature 30Formalin 3-7% with 0-1% Brij 35 Tris, pH 7-6 Room temperature 20Glutaraldehyde -0% 0lM cacodylate, pH 7-4 +40C 15Glutaraldehyde 1-0% with 0 1% saponin 0 IM cacodylate, pH 7-4 + 40C 5, 10, 15Glutaraldehyde 1-0% with 3-7% formalin 0-05M Tris/HCI, pH 7-4 Room temperature 20Glutaraldehyde 1-0% with 3-7% formalin 0-1M cacodylate, pH 7-4 +4°C 15Glutaraldehyde 1-0% with 3-7% formalin PBS, pH 7-4 +40C 1,3, 10Glutaraldehyde 1-0% with 3-7% formalin PBS, pH 7-4 Room temperature 3Bouin's fluid None Room temperature 5 and 10

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1220dilutions of 1/50 or greater. Thus 1/50 was adopted asthe lowest dilution at which guinea pig serum could beused in the system.To examine the effects of fixation of the sections on

specific staining by anti-LSP antibodies, a wide rangeof fixatives and conditions was investigated (table 1).It was found that with the precipitating fixatives,acetone, ethanol, or methanol, some membrane stain-ing with anti-LSP could be obtained but this wasgeneraily weak and the signal: noise ratio was too greatto permit confident interpretation ofstaining patterns.Similarly, paraformaldehyde fixation resulted in onlyweak staining of liver cell surfaces and was associatedwith various artefacts. Among the other crosslinkingfixatives, formalin proved to be fairly effective-resulting in stronger membrane staining than para-formaldehyde, with few artefacts-particularly whenit was buffered with phosphate and at shorter (0 5 to 2minute) fixation times, but when detergents (Brij 35 orTriton X-100) were added to the formalin almost nostaining was obtained with the anti-LSP antisera.

In sections fixed with glutaraldehyde alone therewas no staining of cell membranes by anti-LSP.Addition offormalin to the glutaraldehyde resulted ina fine membrane staining pattern, the intensity ofwhich varied inversely with fixation time but wasindependent of the buffer used. The inclusion ofsaponin (without formalin) in the glutaraldehydeyielded the strongest staining of membranes but somestaining of control sections with normal guinea pigserum (1/50) was also obtained.The most satisfactory result was obtained when

sections were fixed with Bouin's fluid, especially whenexposure to the fixative was kept to five minutes. Clear,strong staining of the sinusoidal portions ofhepatocellular plasma membranes and (to a lesserextent) ofbasolateral and cytoplasmic membranes wasevident (figs 2a, b, and c), at anti-LSP dilutions of 1/50to 1/50 000. In contrast, sections incubated withnormal guinea pig serum at 1/50 dilution werecompletely negative. The sensitivity ofthe procedure isshown by comparison with the radioimmunoassaytitres of the four anti-LSP antisera used (table 2).

Table 2 Comparison of titres obtained byradioimmunoassay and by the ABC immunohistochemicaltechnique withfour guinea pig anti-LSP antisera

Anti-LSP ABC immunohistochemical Radioimmunoassayantiserum titre* titre

GP 9 1/20 000 1/160 000GP 10 1/50000 1/160000GP 11 1/5 000 /100 000GP 12 1/50000 1/220000

*End pint = highest dilution at which a definite positive reactioncould bc detected.

Sipos, McFarlane, Williams, McFarlaneOf the various tissues other than liver that were

examined in this study, positive reactions with theanti-LSP antisera under these conditions were seenonly with kidney. Staining of rat renal tubularepithelium, with prominent epithelial surfacemembrane and weak cytoplasmic staining ofproximaltubular cells, was seen when low dilutions (less than1/400) of the antisera were used. At higher dilutions(1/400 or greater), the antisera stained only liver. Thissuggests that the highest titres of antibodies in theseantisera were directed at liver specific determinants.

Discusson

The present study shows that an enzyme immuno-histochemical technique based on the alkalinephosphatase avidin biotin complex (ABC AP) systemis a reliable method of detecting liver plasmamembrane antigens by specific antisera. The techniquehas sensitivity approaching that ofradioimmunoassayand offers a number of advantages over othermethods. In contrast to indirect immunofluorescence,which-although comparatively insensitive-iswidely used for tissue localisation of antigens, theABC AP system provides a permanent preparation(thereby avoiding the need for photographic records)with well defined morphological details that can beseen by ordinary light microscopy.The inability of other workers6 to detect plasma

membrane antigens with anti-LSP antisera on unfixedcryostat sections of liver is probably the result of acombination of factors including the small amounts ofantigens that are available for interaction with theantibodies and the loss of some of these antigensduring the obligatory washing of the sections. Thenecessity for fixation was shown by Lee et al,78 whoused Bouin's fluid with indirect immunofluorescence,and by Helin et al9 on paraffin wax embedded materialwith an ABC peroxidase system. In the studies of Leeet al78 staining of hepatocellular plasma membranes(with no cytoplasmic staining) was seen with serumsamples diluted up to 1/320, but in that of Helin et al9animal antisera were used only at a dilution of 1/500.Helin et al reported considerable variability inpatterns of staining, including both hepatocellularplasma membrane and diffuse cytoplasmic staining(similar to that shown in fig 1) that made inter-pretation of the results difficult. From the presentfindings it seems that this variability may have beencaused by the fixation conditions, by non-specificinteractions between avidin and the peroxidase orbecause they did not block endogenous biotin beforestaining, or by a combination of these factors.The importance offixation conditions is underlined

in the present study. The precipitating fixatives(acetone, ethanol, or methanol) are generally

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Identification ofliver membrane antigens 1221inadequate for immobilisation of all protein antigensand do not stabilise surface membrane componentssufficiently for immunohistochemical procedures thatentail prolonged incubations or multiple washings ofthe sections. Cross linking fixatives have shown morepromise in this regard but glutaraldehyde, which hasproved useful in enzyme histochemistry, is ofteninadequate for immunohistochemistry'3 and this wasconfirmed in the present study. Addition of formalinor saponin to the glutaraldehyde considerablyimproved stabilisation of the membrane constituents(especially when fixation time was kept short) butsome non-specific staining with normal guinea pigserum was seen and the formalin glutaraldehydesystem was not superior to formalin alone. We confirmthe finding of other studies,'4 that detergents shouldnot be used. A short exposure to Bouin's fluid provedto be superior to all other fixatives and fixationconditions investigated in the present study. As notedby Lee et af Bouin's fluid has the added advantagewhen used for the study of circulating liver auto-antibodies in that it apparently denatures the targetantigens of non-organ specific autoantibodies such asantinuclear, antismooth muscle, and antimitochon-drial antibodies that are common in patients withvarious liver diseases and which would give stainingpatterns on liver sections that could make specificstaining due to anti-LSP antibodies difficult to inter-pret. The mechanism of action of Bouin's fluid isunknown, but the possibility that this denaturing ofcertain proteins (for example, nuclear, smooth muscle,and mitochondrial antigens) may stabilise thehepatocyte surface as suggested by Lee et af seemsuntenable because the anti-LSP antisera were raisedagainst a presumably undenatured antigen prepara-tion. It is unlikely therefore that the antibodies wouldbind to antigens that had been appreciably altered bythe fixative.Although problems of non-specific reactions and

background staining are common with immunohisto-chemical techniques,'0 they were not encountered inthe present study when optimal conditions prevailed.Specific inactivation of endogenous biotin(particularly in liver, which has a high content of thevitamin) is imperative, irrespective of which fixativesor fixation conditions are used. Non-specific bindingof avidin (which has a high isoelectric point, pH 100)to certain classes ofproteins can be a problem and canbe avoided by using the ABC reagent at pH 9.4 (whichdoes not interfere with binding of specificantibodies).5 We could, however, find no differencebetween PBS at pH 7-5 and carbonate buffer atpH 9.4.Endogenous alkaline phosphatase activity iseffectively blocked by including levamisole in thesubstrate buffer, and presumably the acetic acid in theBouin's fluid also contributes to blocking of alkaline

phosphatase isoenzymes.`6 The possibility that guineapig serum glycoproteins might bind to hepatocytes bythe hepatic asialoglycoprotein receptor wasconsidered but such binding requires Ca'+ (which wasnot present in the buffers)," and pretreatment of thesections with a-methyl-D-mannoside to block thereceptor' had no effect on the specific binding of anti-LSP antibodies to the hepatocyte membranes.The ABC system is generally 20 to 40 times more

sensitive than procedures such as the unlabelledperoxidase antiperoxidase method,'8 and the presentstudy indicates that the sensitivity of the ABC APsystem in particular makes it eminently suitable for theidentification of hepatocellular plasma membraneantigens with anti-LSP antisera. In addition to carefulattention to washing protocols and incubation times,the important features of the technique include the useof Bouin's fluid for fixation and the absolute necessityfor blocking endogenous biotin and alkalinephosphatase. Finally, we would recommend theVector Red alkaline phosphatase substrate used in thisstudy in preference to the "black" or "blue" substratesoffered by the manufacturer because the red givessuperior contrast with the counterstainand, unlike the "blue", permits subsequent dehydra-tion and mounting of the sections as permanentpreparations.

JS was supported by the Wellcome Trust.

Reference

1 McFarlane IG. Editorial review: autoimmunity in liver disease.Clin Sci 1984;67:569-78.

2 Meyer zum Buschenfelde KH, Manns M. Mechanisms of auto-immune liver disease. Semin Liver Dis 1984;4:26-35.

3 McCullough A.J. Laboratory assessment of liver function andinflammatory activity in chronic active hepatitis. In: Czaja AJ,Dickson ER, eds. Chronic active hepatitis-the Mayo Clinicexperience. New York: Marcel Dekker, 1986:205-46.

4 McFarlane IG, Williams R. Review: liver membrane antibodies.J Hepatol 1985;1:313-9.

5 Vento S, O'Brien CJ, McFarlane BM, McFarlane IG, EddlestonALWF, Williams R. T-lymphocyte sensitization to hepatocyteantigens in autoimmune chronic active hepatitis and primarybiliary cirrhosis: evidence for different underlying mechanismsand different antigenic determinants as targets. Gastro-enterology 1986;91:810-17.

6 Lebwohl N, Gerber M. Characterization and demonstration ofhuman liver specific protein (LSP) and apo-LSP. Clin ExpImmunol 1981;46:435-42.

7 Lee WM, Shelton LL, Galbraith RM. Antibodies to hepatocytemembrane antigens in chronic liver disease: Detection byimmunofluorescence after Bouin's fixation. J HistochemCytochem 1983;31:1246-9.

8 Lee WM, Martin KL, Shelton LL, Galbraith RM. Hepaticmembrane antibodies: studies ofprevalence and specifcity. ClinExp Immunol 1985;62:715-23.

9 Helin H, Uibo R, Paronen I, Krohn K. Immunohistochemicallocalization ofhuman liver-specific protein using rabbit antiseraand the avidinbiotin complex technique. Clin Exp Immunol1985;59:371-6.

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1222 Sipos, McFarlane, Williams, McFarlane10 Sheibani K, Tubbs RR. Enzyme immunohistochemistry: technical

aspects. Seminars in Diagnostic Pathology 1984;1:235-50.11 McFarlane IG, Wojcicka BM, Zucker GM, Eddlston ALWF,

Williams R. Purification and characterization of human liver-specific membrane lipoprotein (LSP). Clin Exp Immunol1977;27:381-90.

12 Jensen DM, McFarlane IG, Nicholson A, Eddeston ALWF,Williams R. The development of a radioimmunoassay for thedetection ofantibodies to a liver-specific membrane lipoprotein(LSP). J Clin Lab Immunol 1978;1:31-5.

13 Hoopwood D. Theoretical and practical aspects ofglutaraldehydefixation. Histochem J 1972;4:267-75.

14 Goldenthal KL, Hedman K, Chen JW, August JT, WillinghamMC. Post-fixation detergent treatment for immunofluorescencesuppresses localization of some integral membrane proteins.J Histochem Cytochem 1985;33:813-20.

15 Bussolati G, Gugliotta P. Non-specific staining of mast cells

by avidin-biotin-peroxidase complexes (ABC). J HistochemCytochem 1983;31:1419-21.

16 Ponder BA, Wilkinson MM. Inhibition of endogenous tissuealkaline phosphatase with the use of alkaline phosphataseconjugates in immunochemistry. J HIistochem Cytochem1981;29'.281-4.

17 McFarlane IG. Hepatic clearance ofserum glycoproteins. Clin Sci1983;64:127-35.

18 Hsu SM, Raine L, Fanger H. A comparative study of the PAPmethod and avidin-biotin complex method for studyingpolypeptide hormones with radioimmunoassay antibodies. AmJClin Pathol 1981;75:734-8.

Requests for reprints to: Dr IG McFarlane, Liver Unit,King's College Hospital, Denmark Hill, London SE5 9RS,England.

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