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Enhancement of immunohistochemical detection of HIV-1 p24 antigen in brain by tyramide signal amplification
Journal of Virological Methods 67 (1997) 103 – 112
Enhancement of immunohistochemical detection of HIV-1 p24 antigen in brain by tyramide signal amplification Pa´draig M. Strappe a,*, Ting Huei Wang a, Chris Anne McKenzie a, Suzanne Lowrie a, Peter Simmonds b, Jeanne E. Bell a a
Neuropathology Laboratory, Department of Pathology, Uni6ersity of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK b Department of Medical Microbiology, Uni6ersity of Edinburgh, Edinburgh EH8 9AG, UK Accepted 7 May 1997
Abstract Human immunodeficiency virus type 1 (HIV-1) infection of the brain has been demonstrated in formalin fixed, paraffin embedded post-mortem brain tissue (PM) by chromogenic immunohistochemistry for the HIV p24 antigen. The sensitivity of antigen detection is increased significantly by tyramide signal amplification (TSA) compared to the conventional peroxidase labelled Avidin–Biotin complex (ABC) technique. The TSA method also permitted the use of a lower concentration of primary antibody than is conventionally used. Sensitivity was enhanced further by microwave irradiation of the paraffin embedded tissues in citrate buffer. HIV-1 p24 antigen was also detected in PM brain tissue by TSA enhanced immunofluorescence and demonstrated increased sensitivity compared to the conventional immunofluorescence technique with a greatly reduced autofluorescence background. © 1997 Elsevier Science B.V. Keywords: Human immunodeficiency virus type 1; p24 antigen; Immunohistochemistry; Tyramide signal amplification; Brain tissue
1. Introduction
* Corresponding author. Tel.: +44 131 5371976; fax + 44 131 5371013.
Central nervous system (CNS) disease is a common manifestation of HIV-1 infection (Budka, 1991) and can present with a variety of neurological, psychological, and psychiatric disturbances
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(Price, 1994). The presence of HIV-1 in brain tissue has been demonstrated by amplification of extracted DNA by polymerase chain reaction (PCR) (Achim et al., 1994; Dondaldson et al., 1994a), in-situ PCR for proviral DNA (Nuovo et al., 1994; Bagasra et al., 1996), in-situ hybridisation for specific HIV-1 mRNA (Saito et al., 1994; Ranki et al., 1995) and immunohistochemical staining for viral antigen (Wiley et al., 1986). Several HIV-1 proteins have been detected by immunohistochemical methods in paraffin embedded tissue and these include gag p24 (Dondaldson et al., 1994b), envelope gp41 (Tonatore et al., 1994), and nef and tat regulatory proteins (Ranki et al., 1995). Immunohistochemistry for viral antigen is a measure of productive infection and an increase in sensitivity and enhancement of staining would be of great benefit to studies into the pathogenesis of disease. Previously described methods for increasing the sensitivity of HIV-1 antigen detection have included trypsin pretreatment of brain tissue followed by overnight incubation of the primary antibody at 4°C (Falagola et al., 1995), permeabilisation of tissue with Triton X – 100 and proteinase K digestion (Davis et al., 1992) and the use of a 5-layer antibody enhancement technique to improve sensitivity (Esiri et al., 1991). The modified Avidin–Biotin peroxidase (ABC) method (Bedetti, 1985) has been used predominantly to visualise antibody bound to HIV antigen followed by diaminobenzidine (DAB) chromogenic detection. Recently, biotin amplification has been described for lectin and c-fos immunohistochemistry (Berghorn et al., 1994; Adams et al., 1992). Amplification of biotin labelled sites is achieved by the deposition of biotinylated tyramide, catalysed by the horseradish peroxidase enzyme (HRP). This reaction results in the covalent deposition of a biotin label which can be detected using HRP labelled streptavidin or streptavidin labelled with a flurochrome, e.g. Texas red. The end point detection can be with a colour reaction with diaminobenzidine (DAB) or by immunofluorescence. This technique was described previously as catalysed reporter deposition (CARD) and has been used in enzyme immunoassays (Bobrow et al., 1989) the amplification of in situ hybridisation
signals (Kerstens et al., 1995) and in immunohistochemical and western blotting detection of antigen using a primary or secondary biotin labelled antibody. Biotin amplification has demonstrated an increase in signal detection over the conventional peroxidase labelled Avidin–Biotin complex (ABC) and permitted the use of a higher dilution of primary antibody (Berghorn et al., 1994). Formalin fixation and processing of PM brain tissue for paraffin embedding preserves excellent morphology for histology but can affect immunohistochemical detection of antigen. Previously reported methods for the detection of HIV-1 antigen in formalin fixed paraffin embedded tissue have involved pretreatment with enzymes such as trypsin, pepsin and proteinase K (Ranki et al., 1995; Esiri et al., 1991 and Davis et al., 1992). Microwave irradiation has been used to enhance immunohistochemical staining with no loss in morphological detail and a range of antibodies has been documented in which staining in fixed tissue sections has been improved (Cuevas et al., 1994; McQuaid et al., 1995). We now demonstrate the increased sensitivity of HIV-1 p24 antigen detection in paraffin embedded tissue using the DU PONT tyramide signal amplification (TSA) assay compared with conventional ABC detection. This technique combined with microwave irradiation of tissue sections in citrate buffer allows the use of a much lower concentration of the primary monoclonal antibody than was used previously with ABC detection.
2. Methods All patients in this comparative study were infected with HIV-1 and had confirmed HIV antibody and died of AIDS related illnesses. Postmortem body tissue was obtained at autopsy between 24 and 48 h after death. Brain tissue was fixed in 10% formalin for 2 weeks and then processed in a tissue-tek VIP (Miles Scientific) before paraffin embedding. Serial 5-mm sections of frontal lobe tissue were floated onto poly-L-lysine coated slides and incubated at 37°C overnight.
P.M. Strappe et al. / Journal of Virological Methods 67 (1997) 103–112
To compare TSA and ABC detection and the limit of dilution for the primary antibody serial sections were cut carefully from the same tissue block.
2.1. Immunohistochemistry Immunohistochemical studies were carried out using a monoclonal antibody to HIV-1 gag p24 antigen (HIV-1 anti-p24 mouse IgG, DU PONT). The sections were first dewaxed in xylene at room temperature for 20 min and then placed in alcoholic picric acid for 15 min to remove formalin pigment. The sections were then washed in water until clear.
2.2. Microwa6e pretreatment The sections were microwaved in 500 mls of 0.1 M citric acid (pH 6.0) at maximum power setting for 15 min using a 600-W microwave oven. The sections were then removed from the oven and left in the citric acid solution for 20 min at room temperature. The tissue sections were washed in water and endogenous peroxidase was quenched by treatment with 3% H2O2 in water for 10 min. The sections were then washed in water before incubation in phosphate buffer saline (PBS) pH 7.4 containing 0.1% bovine serum albumen (BSA) for 5 min. The slides were next attached to disposable immunostaining chambers (Shandon) and placed in an immunostaining sequenza rack (Shandon). Immunostaining was carried out by the Avidin–Biotin peroxidase complex method and tyramide signal amplification (TSA, DuPont). The following steps were carried out for both TSA and ABC detection: (1) The sections were blocked for non-specific staining with 500 ml of 20% normal rabbit serum (NRS), 0.1% BSA in PBS for 20 min. (2) The primary monoclonal antibody (HIV-1 anti p24 mouse IgG) was diluted in 20% NRS, 0.1% BSA in PBS and 100 ml was applied to the tissue for 30 min at room temperature. (3) The sections were washed with PBS + 0.1%BSA for 3× 5 min.
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(4) The tissue sections were then incubated at room temperature for 30 min with 100 ml of biotinylated rabbit anti-mouse IgG (DAKO), diluted 1:200 in PBS + 20% NRS and 0.1% BSA. (5) The sections were washed with PBS for 3× 5 min.
2.3. Detection by a6idin-biotin complex (6) The HRP labelled Avidin–Biotin complex (ABC) was made according to the manufacturers protocol (DAKO) and 100 ml was applied to the tissue sections and incubated at room temperature for 30 min. The ABC solution was made 30 min before use. (7) Tissue sections were rinsed in PBS and two drops of diaminobenzidine (DAB) solution were applied to each section. Tissue was stained for 2–10 min and then washed in water and counterstained in Mayer’s haematoxylin.
2.4. Detection by tyramide signal amplification (TSA) The detection of HIV antigen by TSA (Du Pont) was carried out according to the manufacturers’ protocol with minor modifications. For TSA detection, steps 1–4 of the ABC method were performed with the same incubation times. Following the incubation of the biotinylated secondary antibody (step 4) the TSA procedure was carried out: (5) The tissue sections were washed for 3× 5 mins with TNT buffer (0.1 M Tris–HCL pH 7.5, 0.15 M NaCl, 0.05% Tween 20). (6) The sections were then blocked for nonspecific binding with 300 ml of TNB buffer (0.1 M Tris–HCl pH 7.5, 0.15 NaCl, 0.5% Du Pont blocking reagent) and incubated at room temperature for 30 min. (7) Then 100 ml of horseradish-peroxidase labelled streptavidin (SA-HRP) diluted 1:500 in TNB buffer was applied to the tissues and incubated for 30 min at room temperature. (8) The tissue sections were washed for 3× 5 mins with TNT buffer.
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(9) For the next step 100 ml of biotinyl tyramide (diluted 1:50 in Du Pont amplification buffer) was applied to the tissue sections and incubated for 10 min at room temperature. (10) The tissue sections were washed for 3× 5 min with TNT buffer. (11) A total of 100 ml of SA-HRP (diluted 1:200 with TNB buffer) was applied to the tissues and incubated for 30 mins at room temperature. (12) The tissue sections were washed for 3× 5 min with TNT buffer and 2 drops of DAB solution was added to each slide and tissue was stained for approximately 30 s and then washed in PBS and counterstained with Mayer’s haematoxylin.
2.5. Con6entional Immunofluorescence detection of HIV p24 antigen. Steps 1–3 of the immunofluorescence procedure were identical to steps 1 – 3 of the ABC/ TSA immunohistochemical method. (4) For this step 100 ml of FITC labelled rabbit anti-mouse immunoglobulin (DAKO) diluted 1:50 in PBS was applied to the tissue sections and incubated for 30 min at room temperature. (5) The tissues were washed for 3 × 5 min with PBS and then mounted with vectashield fluorescent mounting medium (Vector Labs) and viewed under a fluorescence microscope (Leitz) using the appropriate filter.
2.6. TSA immunofluorescence detection of HIV p24 antigen. Steps 1–11 were carried out as described in the TSA immunohistochemical method. (12) Then 100 ml of Texas-Red labelled streptavidin (SA-RED) diluted 1:500 in TNB buffer was applied to the tissue sections and incubated at room temperature for 30 min. (13) The sections were then washed for 3 × 5 min with TNT buffer and then mounted with vectashield fluorescence mounting medium and viewed under a UV fluorescence microscope.
3. Results Microwave irradiation of brain tissue sections in 0.1 M citrate buffer (pH 6.0) caused no impairment of tissue morphology but enhanced the sensitivity of detection of p24 antigen by the ABC method and allowed the antibody to be used at a 1:200 dilution compared to a 1:20 dilution without microwave irradiation. Titration of the p24 monoclonal antibody on serial tissue sections from the same brain block and comparing the TSA with ABC detection systems determined that the optimal dilution of the p24 antibody producing maximum staining was 1:3200 using TSA and 1:200 using ABC (Fig. 1). At antibody dilutions between 1:200 and 1:800 a greater background staining and a reduced specificity was observed with TSA detection. Comparison of the intensity of staining of HIV-1 p24 antigen for TSA and ABC detection methods was demonstrated by immunohistochemistry on serial sections of brain tissue (Table 1). The TSA method gave a consistently more intense staining pattern than ABC in 13 of the 21 brain tissues studied. The intensity of antigen staining was assessed separately in both white and gray matter tissue of the brain by two individuals (PMS and THW), with the white matter showing greater staining. In seven cases that were negative by the ABC method TSA detection demonstrated weak but positive immunohistochemical staining To assess whether the TSA method resulted in an increase in number of p24 antigen positive foci when compared to the ABC method, serial sections were taken from nine cases and immunohistochemistry was performed using TSA and ABC (Fig. 2). The number of antigen positive foci was counted within a 4× 3 inch recticle (Olympus) in two separate areas of the tissue section and by two separate individuals (PMS and THW). An average of the two counts was recorded. To ensure that the same area was examined for both serial sections, foci were counted near a blood vessel or edge that was common to both sections. The TSA method demonstrated at least a two fold increase in the number of antigen positive foci over the ABC method (Table 2) and was statistically significant (PB 0.001).
P.M. Strappe et al. / Journal of Virological Methods 67 (1997) 103–112
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Fig. 1. Comparison of immunohistochemical staining for HIV-1 p24 antigen on serial sections of brain tissue. (A) Detection by TSA with antibody at 1:3200 dilution. (B) Detection by ABC with antibody at 1:200 dilution.
The immunofluorescence detection of p24 antigen using the TSA method with Texas-Red labelled streptavidin enhanced the sensitivity and
also demonstrated a reduced background staining when compared to the conventional immunofluorescence detection. The optimum dilution
Fig. 2. Detection of HIV-1 p24 antigen in serial sections of frontal lobe tissue from two cases (A + B) and (C +D) demonstrating increased sensitivity of antigen detection by TSA. (A +C) p24 antigen detected by TSA, anti p24 antibody dilution 1:3200. (B + D) p24 antigen detection by ABC, anti p24 antibody dilution 1:200.
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of p24 antibody for TSA enhanced immunofluorescence was 1:200 and conventional immunofluorescence was insensitive to antigen detection at this dilution (Fig. 3).
Table 2 Comparison of the number of p24 antigen foci detected by TSA and ABC methods on serial sections of frontal lobe brain tissue Case no.
TSA
ABC
P1
25 21
11 9
P2
47 53
30 17
P4
34 34
26 14
P7
22 38
10 30
P9
15 28
8 8
P11
17 24
8 10
P13
32 24
26 12
P17
22 14
12 8
P22
25 25
16 15
4. Discussion The detection of HIV-1 viral antigen by immunohistochemistry is a rapid and widely used method of determining infection in PM brain tissue. Monoclonal antibodies to HIV-1 gag p24 or envelope gp41 have been used in several studies on paraffin embedded brain tissue with various forms of enzymatic pretreatment (Ranki et al., 1995; Esiri et al., 1991; Davis et al., 1992). The use of a HRP labelled Avidin – Biotin complex to detect the bound antibody with diaminobenzidine Table 1 Comparison of intensity of TSA vs. ABC immunohistochemical detection of HIV-1 p24 antigen on serial sections of frontal lobe tissue Case no.
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21
TSA
ABC
WM
GM
WM
GM
3 2 3 4 0 2 3 2 3 4 4 3 0 4 1 1 3 1 0 0 1
0 1 2 2 1 1 1 3 1 2 4 2 1 3 1 1 1 0 0 1 1
1 1 0 2 0 1 1 2 1 1 1 2 0 2 0 0 1 0 0 0 0
0 0 0 1 0 0 0 0 1 1 1 1 1 1 0 0 0 0 0 0 0
WM, white matter; GM, gray matter. Intensity of staining: 4, very strong; 3, strong; 2, moderate; 1, weak; 0, no staining.
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Statistical analysis: paired t-test PB0.001. Two areas of each section were examined within a defined recticle.
chromogen has also been used in these studies. However there has been no description of HIV-1 antigen detection by immunofluorescence in paraffin embedded tissue, although biotin amplification by deposition of biotinylated tyramide has enhanced c-fos immunohistochemistry and allowed immunofluorescence detection with greatly reduced background (Berghorn et al., 1994). Biotin amplification using the Du Pont TSA reagents with microwave irradiation of tissue sections can be used effectively to enhance immunohistochemical detection of HIV-1 p24 antigen. This was demonstrated by titration of the anti-p24 monoclonal antibody on serial sections of tissue and comparing the sensitivity by detection with TSA and ABC. The use of biotin amplification coupled with HRP staining allowed the antibody to be used at a dilution of 1:3200 compared to a 1:200 dilution
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Fig. 3. Comparison of immunofluorescence detection of HIV-1 p24 antigen in paraffin embedded brain tissue. (A) Conventional immunofluorescence technique for p24 antigen demonstrating insensitivity using an FITC labelled secondary antibody. (B) TSA-Texas red immunofluorescence detection of p24 antigen in paraffin embedded brain tissue. Primary antibody dilution 1:200.
P.M. Strappe et al. / Journal of Virological Methods 67 (1997) 103–112
with ABC detection.This reduction in primary antibody concentration for use in immunohistochemistry has been previously reported for other antibodies (Berghorn et al., 1994). TSA detection also produced a consistently more intense signal with DAB chromogen compared with ABC detection. This significant increase in intensity was demonstrated in 13 of the 21 individual brain tissues examined. Seven tissues showed a weak but positive staining pattern for antigen by TSA and proved negative by ABC detection. The sensitivity of p24 antigen detection by TSA and ABC methods was assessed in serial sections of nine individual brain sections. TSA detection showed a significant increase in the number of antigen expressing foci, up to 2-fold when compared to ABC detection. In this study we have demonstrated that TSA not only increases the intensity of immunohistochemical staining but also numerically detects more HIV-1 antigen expressing cells in brain tissue. The additional p24 positive cells revealed by the TSA method all appear morphologically to be microglia or macrophages as with the ABC method. Clearly TSA enhanced immunohistochemistry will have wide application to studies of low level viral expression or cellular markers that were previously undetected due to the effects of formalin fixation and paraffin embedding on tissue. An important advantage of biotin amplification was seen with immunofluorescence staining. This technique gave an intense fluorescence staining with very low background staining. Previously the conventional immunofluorescence technique for p24 antigen using an FITC labelled secondary antibody could not detect antigen in sections that were positive by ABC detection and some sections demonstrated a high background fluorescence. For TSA immunofluorescence the primary antibody was used at a 1:200 dilution and any further dilution resulted in a reduction in sensitivity. In our study Texas-red labelled HRP was used for immunofluorescence detection, fluorescein and coumarin labelled HRP can also be used in TSA detection which will allow double immunostaining techniques to detect viral antigen and the cell type that is being infected. These approaches are currently being investigated in our laboratory.
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Acknowledgements This work was supported by grant SPG 892571G from the Medical Research Council (UK).
References Achim, C.L., Wang, R., Miners, D.K., Wiley, C.A., 1994. Brain viral burden in HIV infection. J Neuropathol. Exp. Neurol. 53, 284 – 294. Adams, J.C., 1992. Biotin amplification of biotin and horse raddish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 40, 1457. Bagasra, O., Lavi, E., Bobroski, L., Khalli, K., Pestaner, J., Tawadros, R., Pomerantz, R., 1996. Cellular resevoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry. AIDS 10, 573 – 585. Berghorn, K.A., Bonnett, J.H., Hoffman, G.E., 1994. c-Fos immunoreactivity is enhanced with biotin amplification. J. Histochem. Cytochem. 42, 1635 – 1642. Bedetti, C.D., 1985. Immunocytochemical demonstration of cytochrome c oxidase with an immunoperoxidase method. J. Histochem. Cytochem. 33, 446 – 452. Bobrow, M.N., Thomas, D.H., Shaughnessy, K.J., Litt, G.J., 1989. Catalyzed reporter deposition, a novel method of signal amplification. J. Immunol. Methods 125, 279 – 285. Budka, H., 1991. Neuropathology of human immunodeficiency virus infection. Brain Pathol. 1, 163 – 175. Cuevas, E.C., Bateman, A.C., Wilkins, B.S., Johnson, P.A., Williams, J.H., Lee, A.H.S., Jones, D.B., Wright, D.H., 1994. Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. J. Clin. Pathol. 47, 448 – 452. Davis, L.E., Hjelle, B.L., Miller, V.E., Palmer, D.L., Llewellyn, A.L., Merlin, T.L., Young, S.A., Mills, R.G., Wachsman, W., Wiley, C.A., 1992. Early viral brain invasion in iatrogenic human immunodeficiency virus infection. Neurology 42, 1736 – 1739. Dondaldson, Y.K., Bell, J.E., Ironside, J.W., et al., 1994a. Redistribution of HIV outside the lymphoid system with onset of AIDS. Lancet 343, 343 – 382. Dondaldson, Y.K., Bell, J.E., Holmes, E.C., Hughes, E.S., Brown, H.K., Simmonds, P., 1994b. In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop. J. Virol. 68, 5991 – 6005. Esiri, M.M., Morris, C.S., Millard, P.R., 1991. Fate of oligodendrocytes in HIV-1 infection. AIDS 5, 1081 – 1088. Falagola, M.F., Hanly, A., Galvao-Castro, B., Petito, C.K., 1995. HIV infection of Human choroid plexus: a possible mechanism of viral entry into the CNS. J Neuropathol. Exp. Neurol. 54, 497 – 503.
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Kerstens, H.M.J., Poddighe, P.J., Hanselarr, A.G.J.M., 1995. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem. 43, 347–352. McQuaid, S., McConnell, R., McMahon, J., Herron, B., 1995. Microwave antigen retrieval for immunocytochemistry on formalin-fixed, paraffin-embedded post-mortem CNS tissue. J. Pathol. 176, 207–216. Nuovo, G., Gallery, F., MacConell, P., Braun, A., 1994. In situ detection of polymerase chain reaction-amplified HIV1 nucleic acids and tumour necrosis factor alpha RNA in the central nervous system. Am. J. Pathol. 144, 659–666. Price, R.W., Understanding the AIDS dementia complex (ADC). In: Price R.W., Perry S.W. HIV, AIDS and the Brain. Raven Press, New York, 1994, pp. 1-45. Ranki, A., Nyberg, M., Vladimar, Ovod, Haltia, M., Elovaar, I., Raininko, R., Haapasalo, H., Krohn, K., 1995. Abun-
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dant expression of HIV nef and re6 proteins in brain astrocytes in vivo is associated with dementia. AIDS 9, 1001 – 1008. Saito, Y., Sharer, L.R., Epstein, L.G., Michaels, J., Mintz, M., Louder, M., Golding, K., Cvetkovich, T.A., Blumberg, B.M., 1994. Overexpression of nef as a marker for restricted HIV-1 infection of astrocytes in postmortem pediatric central nervous tissues. Neurology 44, 474 – 481. Tonatore, C., Chandra, R., Berger, J.R., Major, E.O., 1994. HIV-1 infection of subcortical astrocytes in the pediatric central nervous system. Neurology 44, 481 – 487. Wiley, C.A., Schrier, R.D., Nelson, J.A., Lampert, P.W., Oldstone, M.B.A., 1986. Cellular localisation of human immunodeficiency virus infection within the brains of acquired immunodeficiency syndrome patients. Proc. Natl. Acad. Sci. USA 83, 7089 – 7093.
Enhancement of immunohistochemical detection of HIV-1 p24 antigen in brain by tyramide signal amplification Pa´draig M. Strappe a,*, Ting Huei Wang a, Chris Anne McKenzie a, Suzanne Lowrie a, Peter Simmonds b, Jeanne E. Bell a a
Neuropathology Laboratory, Department of Pathology, Uni6ersity of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK b Department of Medical Microbiology, Uni6ersity of Edinburgh, Edinburgh EH8 9AG, UK Accepted 7 May 1997
Abstract Human immunodeficiency virus type 1 (HIV-1) infection of the brain has been demonstrated in formalin fixed, paraffin embedded post-mortem brain tissue (PM) by chromogenic immunohistochemistry for the HIV p24 antigen. The sensitivity of antigen detection is increased significantly by tyramide signal amplification (TSA) compared to the conventional peroxidase labelled Avidin–Biotin complex (ABC) technique. The TSA method also permitted the use of a lower concentration of primary antibody than is conventionally used. Sensitivity was enhanced further by microwave irradiation of the paraffin embedded tissues in citrate buffer. HIV-1 p24 antigen was also detected in PM brain tissue by TSA enhanced immunofluorescence and demonstrated increased sensitivity compared to the conventional immunofluorescence technique with a greatly reduced autofluorescence background. © 1997 Elsevier Science B.V. Keywords: Human immunodeficiency virus type 1; p24 antigen; Immunohistochemistry; Tyramide signal amplification; Brain tissue
1. Introduction
* Corresponding author. Tel.: +44 131 5371976; fax + 44 131 5371013.
Central nervous system (CNS) disease is a common manifestation of HIV-1 infection (Budka, 1991) and can present with a variety of neurological, psychological, and psychiatric disturbances
0166-0934/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 0 9 3 4 ( 9 7 ) 0 0 0 8 3 - 9
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(Price, 1994). The presence of HIV-1 in brain tissue has been demonstrated by amplification of extracted DNA by polymerase chain reaction (PCR) (Achim et al., 1994; Dondaldson et al., 1994a), in-situ PCR for proviral DNA (Nuovo et al., 1994; Bagasra et al., 1996), in-situ hybridisation for specific HIV-1 mRNA (Saito et al., 1994; Ranki et al., 1995) and immunohistochemical staining for viral antigen (Wiley et al., 1986). Several HIV-1 proteins have been detected by immunohistochemical methods in paraffin embedded tissue and these include gag p24 (Dondaldson et al., 1994b), envelope gp41 (Tonatore et al., 1994), and nef and tat regulatory proteins (Ranki et al., 1995). Immunohistochemistry for viral antigen is a measure of productive infection and an increase in sensitivity and enhancement of staining would be of great benefit to studies into the pathogenesis of disease. Previously described methods for increasing the sensitivity of HIV-1 antigen detection have included trypsin pretreatment of brain tissue followed by overnight incubation of the primary antibody at 4°C (Falagola et al., 1995), permeabilisation of tissue with Triton X – 100 and proteinase K digestion (Davis et al., 1992) and the use of a 5-layer antibody enhancement technique to improve sensitivity (Esiri et al., 1991). The modified Avidin–Biotin peroxidase (ABC) method (Bedetti, 1985) has been used predominantly to visualise antibody bound to HIV antigen followed by diaminobenzidine (DAB) chromogenic detection. Recently, biotin amplification has been described for lectin and c-fos immunohistochemistry (Berghorn et al., 1994; Adams et al., 1992). Amplification of biotin labelled sites is achieved by the deposition of biotinylated tyramide, catalysed by the horseradish peroxidase enzyme (HRP). This reaction results in the covalent deposition of a biotin label which can be detected using HRP labelled streptavidin or streptavidin labelled with a flurochrome, e.g. Texas red. The end point detection can be with a colour reaction with diaminobenzidine (DAB) or by immunofluorescence. This technique was described previously as catalysed reporter deposition (CARD) and has been used in enzyme immunoassays (Bobrow et al., 1989) the amplification of in situ hybridisation
signals (Kerstens et al., 1995) and in immunohistochemical and western blotting detection of antigen using a primary or secondary biotin labelled antibody. Biotin amplification has demonstrated an increase in signal detection over the conventional peroxidase labelled Avidin–Biotin complex (ABC) and permitted the use of a higher dilution of primary antibody (Berghorn et al., 1994). Formalin fixation and processing of PM brain tissue for paraffin embedding preserves excellent morphology for histology but can affect immunohistochemical detection of antigen. Previously reported methods for the detection of HIV-1 antigen in formalin fixed paraffin embedded tissue have involved pretreatment with enzymes such as trypsin, pepsin and proteinase K (Ranki et al., 1995; Esiri et al., 1991 and Davis et al., 1992). Microwave irradiation has been used to enhance immunohistochemical staining with no loss in morphological detail and a range of antibodies has been documented in which staining in fixed tissue sections has been improved (Cuevas et al., 1994; McQuaid et al., 1995). We now demonstrate the increased sensitivity of HIV-1 p24 antigen detection in paraffin embedded tissue using the DU PONT tyramide signal amplification (TSA) assay compared with conventional ABC detection. This technique combined with microwave irradiation of tissue sections in citrate buffer allows the use of a much lower concentration of the primary monoclonal antibody than was used previously with ABC detection.
2. Methods All patients in this comparative study were infected with HIV-1 and had confirmed HIV antibody and died of AIDS related illnesses. Postmortem body tissue was obtained at autopsy between 24 and 48 h after death. Brain tissue was fixed in 10% formalin for 2 weeks and then processed in a tissue-tek VIP (Miles Scientific) before paraffin embedding. Serial 5-mm sections of frontal lobe tissue were floated onto poly-L-lysine coated slides and incubated at 37°C overnight.
P.M. Strappe et al. / Journal of Virological Methods 67 (1997) 103–112
To compare TSA and ABC detection and the limit of dilution for the primary antibody serial sections were cut carefully from the same tissue block.
2.1. Immunohistochemistry Immunohistochemical studies were carried out using a monoclonal antibody to HIV-1 gag p24 antigen (HIV-1 anti-p24 mouse IgG, DU PONT). The sections were first dewaxed in xylene at room temperature for 20 min and then placed in alcoholic picric acid for 15 min to remove formalin pigment. The sections were then washed in water until clear.
2.2. Microwa6e pretreatment The sections were microwaved in 500 mls of 0.1 M citric acid (pH 6.0) at maximum power setting for 15 min using a 600-W microwave oven. The sections were then removed from the oven and left in the citric acid solution for 20 min at room temperature. The tissue sections were washed in water and endogenous peroxidase was quenched by treatment with 3% H2O2 in water for 10 min. The sections were then washed in water before incubation in phosphate buffer saline (PBS) pH 7.4 containing 0.1% bovine serum albumen (BSA) for 5 min. The slides were next attached to disposable immunostaining chambers (Shandon) and placed in an immunostaining sequenza rack (Shandon). Immunostaining was carried out by the Avidin–Biotin peroxidase complex method and tyramide signal amplification (TSA, DuPont). The following steps were carried out for both TSA and ABC detection: (1) The sections were blocked for non-specific staining with 500 ml of 20% normal rabbit serum (NRS), 0.1% BSA in PBS for 20 min. (2) The primary monoclonal antibody (HIV-1 anti p24 mouse IgG) was diluted in 20% NRS, 0.1% BSA in PBS and 100 ml was applied to the tissue for 30 min at room temperature. (3) The sections were washed with PBS + 0.1%BSA for 3× 5 min.
105
(4) The tissue sections were then incubated at room temperature for 30 min with 100 ml of biotinylated rabbit anti-mouse IgG (DAKO), diluted 1:200 in PBS + 20% NRS and 0.1% BSA. (5) The sections were washed with PBS for 3× 5 min.
2.3. Detection by a6idin-biotin complex (6) The HRP labelled Avidin–Biotin complex (ABC) was made according to the manufacturers protocol (DAKO) and 100 ml was applied to the tissue sections and incubated at room temperature for 30 min. The ABC solution was made 30 min before use. (7) Tissue sections were rinsed in PBS and two drops of diaminobenzidine (DAB) solution were applied to each section. Tissue was stained for 2–10 min and then washed in water and counterstained in Mayer’s haematoxylin.
2.4. Detection by tyramide signal amplification (TSA) The detection of HIV antigen by TSA (Du Pont) was carried out according to the manufacturers’ protocol with minor modifications. For TSA detection, steps 1–4 of the ABC method were performed with the same incubation times. Following the incubation of the biotinylated secondary antibody (step 4) the TSA procedure was carried out: (5) The tissue sections were washed for 3× 5 mins with TNT buffer (0.1 M Tris–HCL pH 7.5, 0.15 M NaCl, 0.05% Tween 20). (6) The sections were then blocked for nonspecific binding with 300 ml of TNB buffer (0.1 M Tris–HCl pH 7.5, 0.15 NaCl, 0.5% Du Pont blocking reagent) and incubated at room temperature for 30 min. (7) Then 100 ml of horseradish-peroxidase labelled streptavidin (SA-HRP) diluted 1:500 in TNB buffer was applied to the tissues and incubated for 30 min at room temperature. (8) The tissue sections were washed for 3× 5 mins with TNT buffer.
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(9) For the next step 100 ml of biotinyl tyramide (diluted 1:50 in Du Pont amplification buffer) was applied to the tissue sections and incubated for 10 min at room temperature. (10) The tissue sections were washed for 3× 5 min with TNT buffer. (11) A total of 100 ml of SA-HRP (diluted 1:200 with TNB buffer) was applied to the tissues and incubated for 30 mins at room temperature. (12) The tissue sections were washed for 3× 5 min with TNT buffer and 2 drops of DAB solution was added to each slide and tissue was stained for approximately 30 s and then washed in PBS and counterstained with Mayer’s haematoxylin.
2.5. Con6entional Immunofluorescence detection of HIV p24 antigen. Steps 1–3 of the immunofluorescence procedure were identical to steps 1 – 3 of the ABC/ TSA immunohistochemical method. (4) For this step 100 ml of FITC labelled rabbit anti-mouse immunoglobulin (DAKO) diluted 1:50 in PBS was applied to the tissue sections and incubated for 30 min at room temperature. (5) The tissues were washed for 3 × 5 min with PBS and then mounted with vectashield fluorescent mounting medium (Vector Labs) and viewed under a fluorescence microscope (Leitz) using the appropriate filter.
2.6. TSA immunofluorescence detection of HIV p24 antigen. Steps 1–11 were carried out as described in the TSA immunohistochemical method. (12) Then 100 ml of Texas-Red labelled streptavidin (SA-RED) diluted 1:500 in TNB buffer was applied to the tissue sections and incubated at room temperature for 30 min. (13) The sections were then washed for 3 × 5 min with TNT buffer and then mounted with vectashield fluorescence mounting medium and viewed under a UV fluorescence microscope.
3. Results Microwave irradiation of brain tissue sections in 0.1 M citrate buffer (pH 6.0) caused no impairment of tissue morphology but enhanced the sensitivity of detection of p24 antigen by the ABC method and allowed the antibody to be used at a 1:200 dilution compared to a 1:20 dilution without microwave irradiation. Titration of the p24 monoclonal antibody on serial tissue sections from the same brain block and comparing the TSA with ABC detection systems determined that the optimal dilution of the p24 antibody producing maximum staining was 1:3200 using TSA and 1:200 using ABC (Fig. 1). At antibody dilutions between 1:200 and 1:800 a greater background staining and a reduced specificity was observed with TSA detection. Comparison of the intensity of staining of HIV-1 p24 antigen for TSA and ABC detection methods was demonstrated by immunohistochemistry on serial sections of brain tissue (Table 1). The TSA method gave a consistently more intense staining pattern than ABC in 13 of the 21 brain tissues studied. The intensity of antigen staining was assessed separately in both white and gray matter tissue of the brain by two individuals (PMS and THW), with the white matter showing greater staining. In seven cases that were negative by the ABC method TSA detection demonstrated weak but positive immunohistochemical staining To assess whether the TSA method resulted in an increase in number of p24 antigen positive foci when compared to the ABC method, serial sections were taken from nine cases and immunohistochemistry was performed using TSA and ABC (Fig. 2). The number of antigen positive foci was counted within a 4× 3 inch recticle (Olympus) in two separate areas of the tissue section and by two separate individuals (PMS and THW). An average of the two counts was recorded. To ensure that the same area was examined for both serial sections, foci were counted near a blood vessel or edge that was common to both sections. The TSA method demonstrated at least a two fold increase in the number of antigen positive foci over the ABC method (Table 2) and was statistically significant (PB 0.001).
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Fig. 1. Comparison of immunohistochemical staining for HIV-1 p24 antigen on serial sections of brain tissue. (A) Detection by TSA with antibody at 1:3200 dilution. (B) Detection by ABC with antibody at 1:200 dilution.
The immunofluorescence detection of p24 antigen using the TSA method with Texas-Red labelled streptavidin enhanced the sensitivity and
also demonstrated a reduced background staining when compared to the conventional immunofluorescence detection. The optimum dilution
Fig. 2. Detection of HIV-1 p24 antigen in serial sections of frontal lobe tissue from two cases (A + B) and (C +D) demonstrating increased sensitivity of antigen detection by TSA. (A +C) p24 antigen detected by TSA, anti p24 antibody dilution 1:3200. (B + D) p24 antigen detection by ABC, anti p24 antibody dilution 1:200.
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of p24 antibody for TSA enhanced immunofluorescence was 1:200 and conventional immunofluorescence was insensitive to antigen detection at this dilution (Fig. 3).
Table 2 Comparison of the number of p24 antigen foci detected by TSA and ABC methods on serial sections of frontal lobe brain tissue Case no.
TSA
ABC
P1
25 21
11 9
P2
47 53
30 17
P4
34 34
26 14
P7
22 38
10 30
P9
15 28
8 8
P11
17 24
8 10
P13
32 24
26 12
P17
22 14
12 8
P22
25 25
16 15
4. Discussion The detection of HIV-1 viral antigen by immunohistochemistry is a rapid and widely used method of determining infection in PM brain tissue. Monoclonal antibodies to HIV-1 gag p24 or envelope gp41 have been used in several studies on paraffin embedded brain tissue with various forms of enzymatic pretreatment (Ranki et al., 1995; Esiri et al., 1991; Davis et al., 1992). The use of a HRP labelled Avidin – Biotin complex to detect the bound antibody with diaminobenzidine Table 1 Comparison of intensity of TSA vs. ABC immunohistochemical detection of HIV-1 p24 antigen on serial sections of frontal lobe tissue Case no.
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21
TSA
ABC
WM
GM
WM
GM
3 2 3 4 0 2 3 2 3 4 4 3 0 4 1 1 3 1 0 0 1
0 1 2 2 1 1 1 3 1 2 4 2 1 3 1 1 1 0 0 1 1
1 1 0 2 0 1 1 2 1 1 1 2 0 2 0 0 1 0 0 0 0
0 0 0 1 0 0 0 0 1 1 1 1 1 1 0 0 0 0 0 0 0
WM, white matter; GM, gray matter. Intensity of staining: 4, very strong; 3, strong; 2, moderate; 1, weak; 0, no staining.
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Statistical analysis: paired t-test PB0.001. Two areas of each section were examined within a defined recticle.
chromogen has also been used in these studies. However there has been no description of HIV-1 antigen detection by immunofluorescence in paraffin embedded tissue, although biotin amplification by deposition of biotinylated tyramide has enhanced c-fos immunohistochemistry and allowed immunofluorescence detection with greatly reduced background (Berghorn et al., 1994). Biotin amplification using the Du Pont TSA reagents with microwave irradiation of tissue sections can be used effectively to enhance immunohistochemical detection of HIV-1 p24 antigen. This was demonstrated by titration of the anti-p24 monoclonal antibody on serial sections of tissue and comparing the sensitivity by detection with TSA and ABC. The use of biotin amplification coupled with HRP staining allowed the antibody to be used at a dilution of 1:3200 compared to a 1:200 dilution
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Fig. 3. Comparison of immunofluorescence detection of HIV-1 p24 antigen in paraffin embedded brain tissue. (A) Conventional immunofluorescence technique for p24 antigen demonstrating insensitivity using an FITC labelled secondary antibody. (B) TSA-Texas red immunofluorescence detection of p24 antigen in paraffin embedded brain tissue. Primary antibody dilution 1:200.
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with ABC detection.This reduction in primary antibody concentration for use in immunohistochemistry has been previously reported for other antibodies (Berghorn et al., 1994). TSA detection also produced a consistently more intense signal with DAB chromogen compared with ABC detection. This significant increase in intensity was demonstrated in 13 of the 21 individual brain tissues examined. Seven tissues showed a weak but positive staining pattern for antigen by TSA and proved negative by ABC detection. The sensitivity of p24 antigen detection by TSA and ABC methods was assessed in serial sections of nine individual brain sections. TSA detection showed a significant increase in the number of antigen expressing foci, up to 2-fold when compared to ABC detection. In this study we have demonstrated that TSA not only increases the intensity of immunohistochemical staining but also numerically detects more HIV-1 antigen expressing cells in brain tissue. The additional p24 positive cells revealed by the TSA method all appear morphologically to be microglia or macrophages as with the ABC method. Clearly TSA enhanced immunohistochemistry will have wide application to studies of low level viral expression or cellular markers that were previously undetected due to the effects of formalin fixation and paraffin embedding on tissue. An important advantage of biotin amplification was seen with immunofluorescence staining. This technique gave an intense fluorescence staining with very low background staining. Previously the conventional immunofluorescence technique for p24 antigen using an FITC labelled secondary antibody could not detect antigen in sections that were positive by ABC detection and some sections demonstrated a high background fluorescence. For TSA immunofluorescence the primary antibody was used at a 1:200 dilution and any further dilution resulted in a reduction in sensitivity. In our study Texas-red labelled HRP was used for immunofluorescence detection, fluorescein and coumarin labelled HRP can also be used in TSA detection which will allow double immunostaining techniques to detect viral antigen and the cell type that is being infected. These approaches are currently being investigated in our laboratory.
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Acknowledgements This work was supported by grant SPG 892571G from the Medical Research Council (UK).
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