J Clin Pathol 1994;47:597-600

Improved double immunohistochemical stainingmethod for cryostat and paraffin wax sections,combining alkaline phosphatase anti-alkalinephosphatase and indirect immunofluorescence

Q Tao, G Srivastava, S L Loke, E Y T Chan, F C S Ho

AbstractAims-To develop an immunohisto-chemical staining method for cryostatand paraffin wax sections so that twodifferent antigens in the same section oftissues could be detected by combiningimmunoenzyme and immunofluores-cence techniques.Methods-This double immunohisto-chemical staining method combinesalkaline phosphatase-anti-alkaline phos-

phatase (APAAP) using New Fuchsin as achromogen and indirect immunofluores-cence.Results-APAAP staining for one antigenof this double immunohistochemicalstaining method was observed underbright field conditions alternating withinimunofluorescence for another antigenunder ultraviolet light. The doubleexposed photograph ofboth easily identi-fied the two signals within the same cell.Conclusions-This double inumunohisto-chemical staining method can overcomethe disadvantages of any masking effectofthe double immunoenzymatic methodsand the background problems of doubleimmunofluorescence method especiallywhen applied to paraffin wax sections. Italso permits good morphological identifi-cation of the doubly stained cells whichmay be of crucial importance in studieson pathology specimens.

(7 Clin Pathol 1994;47:597-600)

Departent ofPathology, UniversityofHong Kong, QueenMary Hospital, HongKongQ TaoG SrivastavaS L LokeE Y T ChanF C S HoCorrespondence to:Dr G Srivastava,Department of Pathology,University of Hong Kong,Queen Mary Hospital,Pokfulam Road, Hong KongAccepted for publication14 December 1993

The double immunohistochemical stainingtechnique is used to demonstrate simultane-ously the presence of two antigens (a) on twodifferent cell types; (b) on different cells of thesame type; and (c) on or inside the same cell.Technically, it is more difficult to performthan single immunostaining because of theneed to avoid cross-reaction and any maskingeffect of signals between the two labelling sys-tems. Several ways of performing doubleimmunohistochemical staining have beenused. Primary antibodies from differentspecies, or primary antibodies of differentimmunoglobulin classes or subclasses fromthe same species can be used, followed by sec-ondary antibodies to species specific or classor subclass specific immunoglobulins labelled

with peroxidase and alkaline phosphatase.Use of directly conjugated antibodies withbiotin or other ligands, followed by strepta-vidin or anti-ligand antibodies conjugatedwith peroxidase or alkaline phosphatase, isanother option. Sequential double enzymaticlabelling using primary antibodies from thesame species was established when it wasrealised that the enzymatic reaction productof the first labelling system can effectivelyblock its own antigenic sites.' 2 Either a com-bined peroxidase and alkaline phosphatasesystem or using the same enzyme system butdifferent chromogens can be used for thedetection step.When the two antigens are located at the

same subcellular site or are very close to eachother-for example, membrane associatedand cytoplasmic-it can be difficult to dis-tinguish between the two enzymatic productsby colour. Detection by double immunofluo-rescence can overcome this defect of detectionby enzymatic reaction products, becausedetection of the signals uses separate filter sys-tems and the two signals do not interfere witheach other. However, this double immunoflu-orescence method is generally more suitablefor studies using cell lines or cell smears whichdo not usually show serious background fluo-rescence, and is less applicable to tissue sec-tions.A double immunohistochemical stain-

ing method, combining immunoperoxidasestaining and indirect immunofluorescence(FITC), has been described by Lechago et alin 1979.3 In this report we describe a doubleimmunostaining system based on the samestrategy but using an improved enzyme anddetection system which can also work onparaffin wax sections.

MethodsCryostat sections (6 pum) were fixed in ace-tone for 10 minutes at room temperature,blocked with 10% normal rabbit serum andincubated with mouse monoclonal antibodies.Normal rabbit serum or irrelevant mono-clonal antibody were used as negative con-trols. The slides were then stained using astandard alkaline phosphatase anti-alkalinephosphatase (APAAP) method.4 Rabbit anti-mouse immunoglobulin and APAAP complex(Dako Japan Ltd) were used at 1 in 50 and 1 in

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100 dilutions, respectively, at 37°C for 30minutes and repeated once. New Fuchsin wasused as a chromogen.The New Fuchsin substrate solution was

prepared fresh just before use by mixing solu-tion I (0 35 ml of 5% New Fuchsin (Fluka72200, in 2M HCI) added to 0-35 ml of fresh4% sodium nitrite (Sigma S-2252) and agi-tated for 60 seconds) with solution II (50 mlof 0-05M TRIS-1 2% NaCl mixed with 18 mlof 2-amino-2-methyl-1,3-propanediol (Merck801464) and adjusted to pH 8-7 to which 28mg Levamisole (Sigma L-9756) was added)to which solution III (35 mg of naphthol AS-BI phosphate (Sigma N-2250) dissolved in0-42 ml N, N-dimethylformamide) was thenadded, and this mixture was used as the NewFuchsin substrate solution.The slides were developed in this freshly

prepared New Fuchsin substrate solution for10 minutes at room temperature, then rinsedin TRIS-buffered saline (0-05M TRIS-0- 15MNaCl, pH 7-6), three times for three minuteseach, and stained by indirect immunofluores-cence method for the second marker (alsousing mouse monoclonal antibody). Wefound that acidic glycine elution5 is not neces-sary before the second step. After incubationwith the second primary antibody FITC con-jugated rabbit anti-mouse immunoglobulinantibodies (Fab'2 fragment) (Dako Japan Ltd)were used at a 1 in 5 dilution for 30 minutesat 37°C. Sections were then mounted with2-5% DABCO/90% glycerol in TBS (pH 8 6)and kept at 4°C in darkness. No counterstain-ing was performed. With serial sectionsstained for the first marker by APAAP, unre-lated antibody of the same isotype as the sec-ond primary antibody was used as a negativecontrol in the second staining step. Positivecells were recorded as single and double expo-sure using a Nikon Optiphot microscope withappropriate filters and Kodak ProfessionalEktachrome EES 800/1600 film (set at ASA800).

ResultsThe results of the double immunohistochemi-cal staining of the cytospin of Raji cell line areshown in fig 1. APAAP staining for B cellmarkers (L26 for CD20) revealed a red colourproduct on Raji cells when observed underbright field conditions (fig lA); indirectimmunofluorescence for Epstein-Barr viruslatent membrane protein (LMP) showed astrong cytoplasmic and weak membraneapple-green fluorescence (fig 1 C); and thedouble exposed photograph of both easilyidentified the two signals within the same cell(fig 1B). The weak cell surface membranestaining seen with antibodies to LMP is pre-sumably due to some masking effect of thisantigen by the alkaline phosphate reactionproduct. The results of double immunohisto-chemical staining identifying the lineage of theLMP positive cells in the cryostat (figs 2A andB) and formalin fixed, paraffin wax sections(figs 2C and D) of an acute lymphoprolifera-tive disorder induced by Epstein-Barr virusare shown in fig 2.

A

B

Figure I Double immunohistochemical staining showingEpstein-Barr virus latent membrane protein (LMP)positive cells in the cytospin preparation ofRap cell line.(A) APAAP stainingfor CD20, pictures taken undertransmitted brightfield illumination; (C) FITCimmunofluorescence showing LMP; (B) double exposureof the left (A) and right (C) column pictures.

DiscussionWe have successfully used this doubleimmunohistochemical staining system todemonstrate the lineage of Epstein-Barr viruspositive cells in lymphomas for one year. It

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Improved double immunohistochemical staining methodfor cryostat andparaffin wax sections

Figure 2 Doubleimmunohistochemicalstaining identifying the Tcell and non-T cell lineageof the LMPpositive cells inthe cryostat (A,B) andformalin fixed, paraffinwax (C,D) sections ofanEpstein-Barr virus induceaacute lymphoproliferativedisorder. Only doubleexposed pictures are shown.For (A) and (B) (arrow):CD3 + LMP + ceUl (A);CD3 - LMP + ceUl (B).Other cells are CD3 +LMP -. For (C) and (D)(arrow):MTI + LMP + cell (C);MTI - LMP + cell (D).Other cells are MTJ +LMP-.

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at the same excitation wavelength as for fluo-rescein isothiocyanate (FITC) fluorescenceand the obvious apple-green colour of FITCstands out of the red background eliminatingany problem created by autofluorescence ofthe section.We found that the APAAP staining for cel-

lular surface markers should be performedfirst, otherwise the enzymatic product ofAPAAP staining for cytoplasmic antigensquenches the antigenic sites of the cellularsurface antigens. Ramshaw and Parums(1992) have also recently described similarstrategy of double immunostaining, but usingFast Red TR salt as chromogen for APAAP.6However, because the enzymatic products ofFast Red give strong red fluorescence underultraviolet light, it can interfere with the FITCsignals.7 We have also found that nitrobluetetrazolium (NBT) can be used as a chro-mogen in our system since it produces nobackground fluorescence as shown by NewFuchsin but the chemical reaction is more dif-ficult to control than with New Fuchsin andhas to be performed in darkness.We recommend the use of this improved

system for double immunostaining for workwhich requires a critical assessment of cellmorphology to identify the type of celllabelled within the heterogeneous cell popula-tion of a tissue section, and where double

has given us constant and reliable results andenabled us to record the morphology of thelabelled cells for detailed analysis. We havealso successfully stained for cell markers andLMP in frozen sections of a lymph nodebiopsy specimen from a patient with acuteEpstein-Barr virus infection and cases of lym-phoma (data not shown) using this method.The pictures of the double exposure of thebright field and fluorescence microscopy eas-ily located the two signals in the same cell.There was no cross-reaction between the twostaining systems. Because the two signals wereobserved using different optical systems, therewas no masking effect with each other. Wehave also successfully stained cryostat sectionsfor bcl-2 oncoprotein (intracytoplasmic) andLMP (mainly cytoplasmic) using this methodon a lymph node biopsy specimen from apatient with acute Epstein-Barr virus infection(results not shown).

Furthermore, we have extended our stain-ing system to formalin and B5 fixed paraffinwax embedded sections. Because formalinand B5 fixed tissue sections usually show seri-ous autofluorescence, double immunofluores-cence can not be applied. However, in ourdouble staining system, after the first APAAPstaining, the enzymatic reaction confers onthe whole section an evenly distributed weakred-colour background under ultraviolet light

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Tao, Srivastava, Loke, Chan, Ho

immunoenzymatic staining cannot be appliedbecause of interference between the twocolour reaction products.

This study was supported by a grant from the University ofHong Kong (CRCG, 337/046/0022). Q Tao is a recipient ofLi Ka Shing Scholarship for postgraduate studies in theUniversity of Hong Kong.

1 Fanili B, Abdulaziz Z, Gerdes J, et al. Description of asequential staining procedure for double immunoenzy-matic staining of pairs of antigens using monoclonalantibodies. Immunol Methods 1986;93:265-73.

2 Wagner L, Worman CP. Colour-contrast staining of twodifferent lymphocyte subpopulations: a two-colour mod-ification of alkaline phosphatase monoclonal anti-alka-line phosphatase complex technique. Stain Technol1988;63:129-36.

3 Lechago J, Sun NCJ, Weinstein WM. Simultaneous visu-

alization of two antigens in the same tissue section bycombining immunoperoxidase with immunofluores-cence techniques. Histochem Cytochem 1979;27:1221-5.

4 Cordell JL, Falini B, Erber WN, et al. Immunoenzymaticlabelling of antibodies using immune complexes of alka-line phosphatase and monoclonal anti-alkaline phos-phatase (APAAP complexes). 7 Histochem Cytochem1984;32:219-29.

5 Bologna M, Allen R, Dulbecco R. A method for doubleimmunofluorescent staining by the indirect procedurewith antibodies of the same isotypes. Immunol Methods1986;86:151-3.

6 Ramshaw AL, Parums DV. Combined immunohistochem-ical and immunofluorescence method to determine thephenotype of proliferating cell populations. Clin Pathol1992;45:1015-17.

7 Murdoch A, Jenkinson E, Johnson GD, Owen JJT.Alkaline phosphatase-Fast Red, a new fluorescentlabel-Application in double labelling for cell surfaceantigen and cell cycle analysis. Immunol Methods1990;132:45-9.

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