800 J Clin Pathol 1990;43:800-805 Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by J Clin Pathol: first published as 10.1136/jcp.43.10.800 on 1 October 1990. Downloaded from in situ hybridisation R G Morris, M J Arends, P E Bishop, K Sizer, E Duvall, CC Bird Abstract ing of streptavidin to endogenous sites within The sensitivity of digoxigenin and biotin tissues.'3 '4 As a putatively more sensitive labelled DNA probes for the detection of alternative, digoxigenin, a derivative of the human papillomavirus (HPV) by dot cardiac glycoside digoxin, has recently been blotting and in situ hybridisation was used to label DNA probes for the successful compared in tissues from cervical, identification of HPV in HeLa and SiHa laryngeal, and anogenital neoplasia. cells."5 Probes were either labelled with digox- igenin by the random primer technique and detected with anti-digoxigenin Methods antibody, or labelled with biotin by nick TISSUE PREPARATION translation and detected with strep- Procedures were adapted from the method of tavidin, both methods having a common Lewis et al.'2 Paraffin wax sections (5 gm) final visualisation procedure using alk- were prepared from routinely processed aline phosphatase. Digoxigenin labelled paraffin wax blocks and placed on 3-amino- probes proved two to 10-fold more sen- propyltriethoxysilane (TESPA; Sigma) coated sitive by quantitative dot blotting and slides'6 and incubated at 42°C for three days four-fold more sensitive in detecting before dewaxing in xylene at 37°C for 30 HPV 16 DNA in a series of 31 anal car- minutes and xylene at room temperature for cinomas, compared with biotinylated 10 minutes. Tissue sections were hydrated probes. The digoxigenin method also through absolute ethanol twice for five min- produced less non-specific background utes, 95%, 85%, 70%O and 50% (v/v) ethanol staining of tissue sections than biotin for two minutes each, and phosphate buffered labelled probes. saline (PBS) for five minutes. They were then It is concluded that digoxigenin DNA immersed in 0-02M hydrochloric acid for 10 labelling and detection provides a sim- minutes, washed in PBS twice for five min- ple, reliable, and efficient alternative to utes, placed in 0.1% (v/v) Triton X-100 in http://jcp.bmj.com/ the use of biotin or radioactive isotopes PBS for one and a half minutes, washed in for the detection of HPV DNA by in situ PBS twice for five minutes, and digested with hybridisation. Digoxigenin labelled 250 Mug/ml proteinase K (Gibco-BRL) in 50 probes also offer the possibility of double mM TRIS (pH 7 6), 5 mM EDTA for 10 labelling in situ hybridisation pro- minutes at 37°C. Further digestion was prevented by washing the slides twice in PBS cedures when used with biotin labelled on October 6, 2021 by guest. Protected copyright. probes to provide simultaneous identi- containing 2 mg/ml of glycine for five min- fication of different DNA sequences. utes, and endogenous alkaline phosphatase activity was inhibited by immersing in 20% (v/v) acetic acid at 4°C for 15 seconds. This Human papillomavirus (HPV) infection has was followed by two 10 minute washes in been strongly implicated in the development PBS, post-fixation in 4% (w/v) paraformalde- of cervical, laryngeal, and anogenital neo- hyde in PBS for five minutes, two five minute plasia, with low grade tumours usually washes in PBS, and dehydration through Department of associated with HPV types 6b and 11, and graded alcohols, as above, to absolute ethanol Pathology, University high grade with HPV types 16 and 18.'-3 where the slides were stored awaiting Medical School, Teviot Place, Although several methods are available for hybridisation. Edinburgh EH8 9AG HPV detection," in situ hybridisation offers For the detection of highly repetitive R G Morris the advantage of cellular localisation of viral sequences, human cells from male buccal M J Arends K Sizer DNA within tissue sections and it can also be mucosa were smeared on to glass microscope E Duvall used with routinely collected paraffin wax slides and allowed to air dry for one minute CC Bird embedded material, permitting retrospective and treated as described below for the detec- Genitourinary investigations. tion of the Y chromosome."' Medicine Unit, Of the of in situ tech- Frozen sections (5 pm) were placed on Department of variety hybridisation Medicine, Royal niques available, radioactively labelled probes, TESPA coated microscope slides and fixed in Infirmary of although highly sensitive, have the disadvan- 4%O paraformaldehyde in PBS for 16-24 Edinburgh tage of safety hazards associated with ionising hours, washed twice in 0 1M TRIS (pH 7-4) P E Bishop radiation and long autoradiography exposure for five minutes, immersed twice in 0 25% Correspondence to: P40 Dr R G Morris times of up to 30 days.'0 The use of biotin as a (v/v) Triton X-100, 0-25% (v/v) Nonidet Accepted for publication non-radioactive label has proved popular" 12 in 0O1M TRIS for five minutes, and washed 15 May 1990 despite the disadvantage of non-specific bind- twice in 0 1M TRIS for five minutes. Slides Sensitivity of digoxigenin and biotin labelled probesfor detection of human papillomavirus by in situ hybridisation 801 were subsequently incubated at 37°C with (w/v) SDS twice for 15 minutes at 65°C. In 100 pg/ml of proteinase K in 50 mM TRIS, some experiments labelled probe was spotted 5 mM EDTA for 10 minutes, washed in 0 1M directly on to nitrocellulose and baked under TRIS containing 2 mg/ml glycine twice for vacuum at 80°C for two hours. J Clin Pathol: first published as 10.1136/jcp.43.10.800 on 1 October 1990. Downloaded from five minutes and immersed in 20% (v/v) acetic acid at 4°C for 15 seconds before being IN SITU HYBRIDISATION washed twice in 0-1M TRIS for five minutes. Hybridisation mixture (50 pl) containing the Slides for subsequent hybridisation with labelled DNA was placed on the prepared biotin-labelled probes had endogenous avidin/ sections, covered with Gelbond (ICN), and biotin binding sites blocked using the recom- the edges sealed with nail varnish. Probe and mended protocol described by the supplier cellular DNA were denatured together by (Vector Laboratories). Briefly, the slides were heating to 90'C for 10 minutes and the slides placed in a humidified box and sections transferred to a humidified box at 42°C for 16 covered with avidin for 20 minutes before hours. After removal of the Gelbond the slides washing twice in 0 IM TRIS for five minutes. were washed at high stringency in 2 x SSC at The slides were returned to the humidified room temperature for 10 minutes, 2 x SSC box and sections covered with biotin for 20 at 60°C for 20 minutes, 0-2 x SSC at room minutes and washed twice in 01M TRIS for temperature for 10 minutes, and 0-2 x SSC at five minutes. All slides were then immersed in 42°C for 20 minutes. 2 x SSC (0-3M sodium chloride, 30 mM sodium citrate at pH 7-0) for 10 minutes and DETECTION OF HYBRIDISED PROBE 2 x SSC, 50% deionised formamide for 60 Both the slides and the dot blots were pro- minutes. cessed at room temperature using the com- mercially available detection kits for use with PREPARATION OF DNA PROBES biotin (Gibco-BRL) or digoxigenin (Boeh- Plasmids containing HPV 11 or 16 cloned into ringer), with slight modifications to the pBR322 (donated by Dr L Gissmann, Heidel- recommended protocols. berg) or human Y chromosome sequences After washing for five minutes in buffer 1, cloned into pBR328 (pHY2- 1) (from Dr HJ containing either 0 1M TRIS (pH 7 5), 0 1M Cooke, Edinburgh) were labelled with either NaCl, 2 mM MgCl2, 0-05% (v/v) Triton X-100 biotin- 11 -dUTP by nick translation'8 or (biotin) or 01M TRIS pH 7*5, 0-15M NaCl digoxigenin- 1 1 -dUTP by random primer (digoxigenin) the slides were immersed in labelling using commercially available kits buffer 1 containing 30% (w/v) bovine serum (Gibco-BRL, Boehringer) following the albumin (biotin) or 20% sheep serum (digox- recommended protocols.'>" DNA (1 Mg) was igenin) for 30 minutes. Slides were then labelled with biotin. With digoxigenin either 1 incubated in a humidified box with buffer 1 pg (dot blots) or 0-2 pg (in situ hybridisation containing either 2 pg/ml of streptavidin and dot blots) were labelled. In some (biotin) or a 1 in 5000 dilution of anti-digox- experiments DNA was labelled with digox- igenin antibody for 20 minutes before being http://jcp.bmj.com/ igenin by nick translation. Before random washed in buffer 1, twice for 20 minutes. Biotin primer labelling the DNA was linearised with slides were returned to the humidified box, the restriction enzymes Bam Hl (HPV 11 and covered in buffer 1 containing 1 pg/ml of 16) or EcoRI and Pvu II (pHY2-1). Unin- biotin-polyalkaline phosphatase for 20 min- corporated nucleotides were removed using utes, before washing in buffer 1, twice for 20 Geneclean (Stratech). For in situ hybridisa- minutes. All slides were then placed in buffer 2 tion, DNA probes were prepared at a concen- containing 01M TRIS (pH 9-5), 01M NaCl, on October 6, 2021 by guest. Protected copyright. tration of 200 ng/ml (biotin) or 140 ng/ml 5 mM MgCl2 for 60 minutes to raise the (digoxigenin) in hybridisation buffer contain- pH. Visualisation reagent containing buffer ing 2 x SSC, 50o (w/v) dextran sulphate, 2, nitro-blue tetrazolium salt (0 33 mg/ml), and 5000 deionised formamide, and 0-2% (w/v) 5-bromo-4-chloro-3-indolyl phosphate (0-17 low fat skimmed milk.