Copyright
Exn Dermaiol 1992: I: 230- 235 Frinied in Denmark . Ail rif^lils reserved
ic) Miinksgiiard
1992
Experimental Dermatotogy ISSN 0906-6705
A non-radiolabelled in situ hybridization method for the detection of epidermal cytokine mRNA Howie SEM, Aldridge RD, McVittie E, Thorntoti E, Ratnage E, Hunter JAA. A noti-radiolabelled /« .V//M hybridization method for the detection of epidertnal cytokine tnRNA. Exp Dermatol 1992: 1: 230-235. © Munksgaard, 1992
S. R. E. J.
E. M. Home\ D. Aldridge', E. McVittie", Thornton\ E. Ramage^ and A. A. Hunter'
Departments of 'Dermatology and ^Pathology, University of Edinburgh, Edinburgh, Scotland
Key words: cytokine mRNA - FITC-labelled oligonucleotide probes
Abstract: We describe a non-radiolabelled method lor the iti .situ detection of epidertnal cytokine mRNA in paraffm sections of skin biopsies frotn sites of nickel contact sensitivity. The method uses Huorescein isothiocyanate-labelled oligonucleotide exon probe cocktails.
Introduction
The need to identify cells within a tnixed cell population containing a nucleic acid of interest led, in the late 196O's, to the development of in situ hybridization (ISH). The technique involves the annealing under stringent conditions of a complementary probe to cellular DNA or RNA. Sensitivity depends upon effective preservation of the target nucleic acids, specificity of the probe, tnaxitnization of hybridization and an efficient detection system. Probe technology has been revolutionized with the production of oligonucleotide probes. Such probes can be tailored to the gene under investigation and made long enough to reduce non-specific interactions yet short enough to penetrate tissues. Furthermore, as avidity can vary between exon sequences, 'cocktails' of probes to different sequences of nucleic acids within the gene of interest can be produced. Recently, exon cocktail oligonucleotide probes to tnessenger RNA sequences have becotne commercially available. Such probes can be labelled at either the 5' or the 3' end depetiding on the enzytne system employed. 230
Dr, S, E, M, Howie, Department ot Pathology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland, Tel, 031 650 2955, Accepted tor publication 24 November 1992
Traditionally, probes have been labelled with radioisotopes, but to shorten detection titne with itnproved resolutioti atid to reduce the health hazard we chose to have the probes labelled with tluorescein isothiocyanate (FITG). The FITC-labelled probes used in this study did not provide sufficient signal for direct visualization by UV tnicroscopy and an itnmunohistochemical detection tnethod was employed. Unlike biotin, FITC gives minitnal background for itnmunohistochetnistry as it does not occur in mammalian tissues. Material and methods
Subjects Five females and 1 tnale fully-informed nickel-sensitive volunteers (age range 24 52 years) each of whotn had a positive patch test (2+ positive; International Cotitact Dertnatitis Research Group scale) (1) within the 2 months preceding the study were recruited. All had 25 mg 5"/» nickel sulphate in white soft paraffin (WSP) applied under 8 tntn Finn chatnbers to three sites on the lower abdotnen which had not previously been affected by eczetna. Patches were retnoved at 3 hours after application and 6 mm punch biopsies taken from the center of
Non-radiolabelled /// situ hybridization
the site after infiltrating the surrounding area with 2 % Lignocaine (Astra). Probes Exon cocktail FITC-labelled oligonucleotide probes, for interleukin-la, interleukin-8, and Actin were purchased frotn British Biotechnology, Cowley, Oxford. Ten-microliter aliquots at 20 ng/|.tl were stored at - 7 0 C in autoclaved disposable plastic Eppendorf tubes until use. Sample preparation T h e biopsies were immediately placed into buffered formalin-saline, and embedded in paraffin at 38 C. Eive-tnicron sections were lloated onto sterile 0.1% diethyl pyrocarbonate (SIGMA, UK) water ( D E P C water; 1 tnl DEPC/liter of deionized glass distilled water, shaken until dissolved and autoclaved) and taken onto 3-aminopropyltriethoxysilane ('Tespa') (Sigma) coated slides. Sections were dried overnight at 56 C and stored in dust-free conditions until used. Sections were dewaxed and rehydrated through clean xylene, and ethanol washes, followed by washing in sterile DEPC water. Sections were incubated in 2X statidard sodium citrate (SSC) with a final rinse in sterile D E P C water. Laboratory preparation for in situ hybridization To preserve RNase-free conditions, solutions were made up in 0.1% DEPC water. All glasswear was heated at 250X for 12 h. All disposable plastics were autoclaved before use. Both glassware and plastics were stored dust-free until use. Gloves were worn at all titnes when handling reagents, glassware or plastics.
Mannheim) in 50 mM Tris-HCl buffer pH 7.6 at 37 C for 60 min enhanced hybridization. The reaction was terminated by washing in phosphate-buffered saline (PBS) and fixing with paraformaldehyde. Preh vbridization, hybridization and posthybridization washes After prelitninary studies exatnining the effects of varying the constituents of the hybridization buffer tnixture (HBM), the cotnposition of HBM which gave the most satisfactory results was established and is shown in Table 1. All reagents were supplied by Sigtna and, where possible, were of molecular reagent grade. Slides bearing single sections were ptehybridized with 60 |il HBM/section and incubated in a humid chamber for 60 tnin at 37 C. Prior to hybridization, HBM was drained off the slide. The labelled probe was then added at various concentrations in HBM. In this study each probe was used at a concentration of 20 ng probe/ 10 pl HBM/section. Gelbond (Flowgen Instrutnents Ltd., England), cut to shape, was placed onto each section. Overnight iticubation at 37 C in a sealed hutnid container was followed by several washes of SSC of decreasing ionic sttength. Probe detection, enhaitcenient and visualization Experiments to detect the probe were undertaken with three monoclonal and one polyclonal antiFITC antibodies. DAKO polyclonal rabbit antiFITC (V403) was the preferred antibody as there was minitnutn background staining. Before use, one part of the antibody was absorbed overnight at 4 C with one part of swine .serutn, one part of whole hutnan blood, and a finely cut preparation of 500 tng whole human skin. The absorbed antibody was then ultracentrifuged and affinity-puri-
Slide preparation To ensure tissue sections retnained on the slides during the prolonged ISH procedure, tnictoscope slides were prepared in the following tnanner. They were soaked in 1% Decon for 1 h, washed in running tap water for a tninitnutn of 30 tnin. After washing in 4 changes of distilled water, they were taken up through 157« ethanol, industrial methylated spirit, 95% ethanol and then air-dried. Each slide was dipped individually in freshly prepared 2 % 'Tespa' in acetone, washed in 2 rinses of DBPC water, air-dried and stored in dust-free conditions. Pretreatnients Prelitninary work showed that incubation of sections with 1 |.ig/ml proteinase K (Boehringer
Table 1. Hybridization Buffer Mixture Constituents
Concentration
Sodium chloride
0.6 M
Sodium pyrophosphafe
0,1%
PVP
0,2%
RGOII
0,2%
EDTA Tris-HCl pH 7,6
5 mM 50 mM
10%
Dextran sulphate DNA (sheared salmon sperm)
10%
'Formamide (deionised)
50%
' Formamide was deionised with mixed bed resin [Sigma M8032] tollowing
the insttucfion sheet
231
Howie et al.
fied on a Protein G column (Phartnacia), in accordance with the tnanufacturer's protocol. Sections were incubated with purified anti-FITC antibody, washed and then, following further incubation with biotinylated anti-rabbit itnmunoglobulins [swine F(ab)n fraction, DAKO E 431], a sensitive streptavidin and biotinylated alkaline phosphatase system was used to detect the hybridization signal [in situ Hybridisation Detection System K 600, DAKO]. This results in an insoluble blue chromogen fortned by the hydrolysis of 5bromo-4-chloto 3 indolyl phosphate (BCIP) and concotnitant enzyme reduction of nitro blue tetrazolium (NBT) at the site of hybridization. Visualization was not improved by counterstaining. Sections were covered with Cristal/tnount (Biogenesis, Bournemouth, England). The final working protocol is given in Table 2. Controls Sections were processed as above with otnission of probe, or with RNase [SIGMA Type 1-A] digestion, 1 mg/ml at 37 C for 60 min prior to prehybridization. Assessment of ISH Microscopic assessment of sections was undertaken by an observer (S.E.M.H.) who was blind to the clinical or the laboratory hybridization procedutes. The effects of many variations on the protocol were examined during development of the tnethod. Results
The protocol given in Table 2 was that which gave the tnost satisfactory results. Figs. 1 and 2 illustrate representative results obtained frotn the 6 nickel sensitive patients included in this study. Fig. 1 shows the hybridization obtained with IL8 probe and shows the effect of pretreatment with RNase illustrating the specificity of the reactions for mRNA. Fig. 2 shows the patterns of hybridization seen in a different patient with probes for IL-l a, IL-8 and actin. Discussion
Messenger RNA has a short life due to the ubiquitous presence of endogenous ribonuclease, an enzytne particularly tesistant to degradation. Thus ISH is an exacting technique requiring an RNasefree environtnent. fn these studies the biopsy was rapidly placed into ]{)'%, bulTered formol saline and 232
Table 2, In Situ Hybridization Protocol Stock Solutions 20XStandard saline citrate (SSC), 3 M NaCl, 0,3 M citric acid 10x Phosphate buffered saline (PBS), 1,3 M NaCl, 70 mM NaHjPO,,, 30 mM NaH^PO, 2H 0 Profeinase K, 1 mg/ml in 50 mM Tris-HCl pH 8,0 containing 1 mM CaCI Stored at 4''C, lOxTris-HCI, 0,5 M, pH 7,6, Tris buffered saline (TBS): 50 mM Tris-HCl, 150 mM NaCl, pH 7,6, Ail procedures are carried out at room temperature, unless otherwise stated uewaxitig and Rehydration 1. Xylene 2. 100% ethanol 3. 95% ethanol 4. 70% efhanol 5, 2XSSC (prewarm to 60°C) 6, DEPC wafer Proteinase K Digestion 1, I X Tris-HCl 2, Proteinase K: 1 ),ig/ml in 1 x Tris-HCl 3, ixPBS 4. 0.4% paratormaldehyde in 1 x PBS 5, DEPC water
2x5 1x1 1x1 1x1
mins min min min
1x10 min at 60°C 1x1 min 1 x 5 mins 1x60 mins at 37 °C 1 x 5 mins 1x20 mins at 4°C 1 x5 mins
RNase Digestion Prepare RNase: 100 mg/ml in 2xSSC, 10 mM MgClj 1, Apply RNase to each section 1 x60 mins at 37 °C 2, 0,4% paraformaldehyde in 1 xPBS 1 x20 mins at 4''C 3, DEPC water 1 x 5 mins 4, Air dry Sections NCT tor RNase treatment: 1, 2XSSC, 10 mM MgCI^ 1 x60 mins at 37 °C Postfix, wash and dry as for RNase digestion Pretiybridization 60 |.il HBM/section
1x60 mins at 37°C
Hybridization Lcnfrol sections: 10 pi HBM/section Test sections: - 2 0 ng probe/10 pl HBM/section Post Hybridization Washes Float oft cover slips in 2xSSC Prewarm SSC solutions at 37 °C 1, 2XSSC 2, 1XSSC 3, 0,5 X SSC 4, 0.2 X SSC 5, TBS with 2 mM MgClj, 0,1% BSA, 0,1% Triton 100
37°C overnight 37 °C overnight
2 x 5 mins 37°C 2 x 5 mins 37°C 2 x 5 mins 37°C 2 x 5 mins 37°C 1x15 mins
Detection Procedure All reagents were diluted in and all washes were carried out in TBS with 0,1% BSA 1, 20% swine serum 1 X10 mins 2, Affinity purified rabbit anfi FITC (1:100) 1x30 mins 3, Rinse each section and wash 3x2 mins 4, Biotinyiafed anti-rabbit Igs (1:500) 1x30 mins 5, Rinse each section and wash 3 x 2 mins 6, 'Streptayidin 1x30 mins 7, Rinse each section and wash 3 x 2 mins 8, "Biotinylated Alkaline Phosphate 1 X30 mins 9, Rinse each section and wash 3x2 mins 10, Distilled water 1 x2 mins 11, *BCIP/NBT substrate 1 x60 mins in dark 12, Distilled water 1 x5 mins 13. Mount in Cristal/mount * From DAKO in situ hybridisation system [K600], as per schedule.
Non-radiolabelled in situ hybridization -»>>**
-C Figure I. In situ liybridiz;ition demonstrating epidermal cytokine mRNA in nickel-challenged skin of a nickel-sensitive subject, (a) no probe, (b) IL-S iiiRNA expression, note the cytoplasmic signal and the absence of nuclear staining, (e) absence of IL-8 niRNA expression rollowing RNa.se digestion. Original magnification x 200.
sections were post-fixed in 0.4'Xi parafortnaldehyde following proteinase K digestion. Both fixatives penetrate tissue rapidly, inactivate RNase and preserve tissue tnorphology. RNA is proteinase-resistant and it has been shown that in ISH, particularly where fixed rather than frozen sections are used, proteinase digestion increases the signal to noise ratio (2). No signal
•%
Fii;i(re 2. Hybridization patterns seen in a further subject, (a) no probe, (b) IL-la mRNA expression, (c) IL-S mRNA expression, aclin niRNA expression. Note the variation of slaining pattern with dilTercnt probes. Original magnification x 100,
233
f
Howie et al.
was detected in our systetn if proteinase K pretreatment of sections was otnitted. This is analogous to the pretreatment of sections with trypsin to reveal certain antigenic moieties in immunohistochemical procedures. However, this enzymatic digestion can increase non-specific adherence of either the probe or the imtnunohistochetnical detection system and thus background staining (3). Tn our studies increasing proteinase K concentration was accompanied by increasing non-specific nuclear staining. Although it is reported that proteinase K increases signals by partially removing the cellular proteins that itnpair probe access (4), it is also possible that it increases tissue valency which, in turn, may increase non-specific probe adherence by electrostatic attraction. Many non-radioisotopic methods have been used to label probes (5), but their sensitivity, particularly in paraffin sections, has been questioned (6). Reeently, a non-radioisotopic ISH technique has been shown to be highly sensitive provided the system is optitnized and sufficiently stringent conditions are employed to tninimize non-specific binding (6). Digoxigenin-labelled probes have been used previously in skin (7), but non-specific adherence has been teported in other tissues (8). In our studies we chose to employ an FITC label as this hapten is not naturally found in tnammalian tissues and thus its detection ought not to be accotnpanied by excessive endogenous background. Initially, amplification was attetnpted by using several antiFITC monoclonal antibodies. The results were disappointing and so we opted to use an anti-FITC polyclonal antibody. Although this resulted in a much enhanced signal it was accompanied by increased non-FITC specific staining. This background was successfully retnoved by absorption of the polyclonal anti-FIT'C with a variety of tissues including skin, followed by affinity purification on protein G. Atnplification of the signal obtained with the anti-FITC antibody was initially attempted with an avidin bridge and detected using Fast Red or Fast Blue as the chromogen for biotinylated alkaline phosphatase but this proved ineffective. Acceptable signal detection was consistently achieved with the more sensitive strepavidin and BCIP/NBT procedure. Messenger RNA degrades with increasing temperature atid alkalinity and it has been foutid that the addition of formamide enables the optimal temperature of annealitig to be reduced. It is also known that the presence of sodium chloride will inlluence annealing by stabilizing the complex. In our studies, satisfactory results were obtained when 50"/) fortnatnide was used with 0.6 M sodium chloride at pH 7.6 at 37 "C overnight. 234
Following hybridization under low-stringency conditions, washing in conditions of high stringency, low salt and high temperature will remove probes with mismatched sequences. In our studies prolonged washing with decreasing ionic coticentrations of SSC tnarkedly reduced background staining. The concentration of the probe used is critical in tnaxitnizing hybtidization. Dextran sulphate is added to the probe mixture; it appears to act as a phase separator as its tnolecular weight prevents it entering the cell and thus effectively raises the concentration of probe in the tissue-diffusible mixture (9). In our studies the addition of dextran sulphate was found to enhatice probe visualization without increasing background. Non-specific binding of probe to cellular tnRNA would be expected to have resulted in identical hybridization patterns regardless of the probe used, as the same amount of DNA was applied to each section. Fig. 2 illustrates that different probes produee quite distinctive staining patterns, indicating that the binding detected is due to differences in the distributions of the mRNAs tested. Positive ISH for cytokine mRNA in the epidermis of nickel-sensitive subjects following antigeti exposure indicates that transcription has occurred but does not confirm the production of protein in significant quantities (translation). Additionally, the technique at ptesent is not atnenable to fortnal quantification. Nevertheless, despite these inherent disadvantages ISH is a powerful tool and unlike Northern blotting allows an individual cell producing specific tnRNA to be identified within tissues. In summary, we report a successful method of detection of cytokine mRNA by non-radiolabelled in situ hybridization. We are currently using this protocol to study iti detail the immediate transcription of cytokine genes in contact sensitivity. The results of these studies will be reported elsewhere.
References 1. Fregert S. Patch testing. In: Manual of Contact Dermatitis. Copenhagen: Munksgaaid, 1981: 71-81. 2. Angerer L M, Angerer R C, Detection of poly A-t-RNA in .sea urchin eggs and embryos by quantitative in situ hybridisation. Nucleic Acids Research 1981: 9: 2819-2840. 3. Curran R C. Gregory J. The unmasking of antigens in paralTm sections of tissues by trypsin. Expetentia 1977: 33: 1400-1401. 4. Naylor M S, Balkwill F R. Northern analysis and //; situ hybridisation of eytokinc mRNA, In: Balkwill F R. ed, Cytokines. Oxford: IRL Oxford Llniveisity Press. 1991: 31 50. 5. Holler H. Principles of in situ hybridisation, ln: Polak J M, McGee J O'D, ed. //; situ Hybridi.sation Principles and Practice. Oxford: Oxford University Press. 1990: 15-29. 6. Fleming K A, Evans M, Rylcy K C, Franklin D, Lovell-
Non-radiolabelled in situ hybridization Badge R H. Morey A L. Optimisation of non-isotopie in .situ hybridisation on formalin-fixed, paraffin-embedded material usine digoxigenin-labelled probes and transgenic tissue J Patholouy 19'92: 167: 9 17, 7 Fisher C. Angus Ii. Rees J. //; siiu hybridisation using digoxigenin labelled probes in human skin, Br.l Derm 1991: 125: 516-520.
8. Mitchell B S, Dhami D. Schumacher U. //; ,s//i( hybridisation: a review of methodologies and applications in the biomedical sciences. Mcd Lab Sci 1992: 49: 107-118, 9. Warlord A. In situ hybridisation: a new tool in pathology. Med Lab Sci 1988:45:381-394,
235
Exn Dermaiol 1992: I: 230- 235 Frinied in Denmark . Ail rif^lils reserved
ic) Miinksgiiard
1992
Experimental Dermatotogy ISSN 0906-6705
A non-radiolabelled in situ hybridization method for the detection of epidermal cytokine mRNA Howie SEM, Aldridge RD, McVittie E, Thorntoti E, Ratnage E, Hunter JAA. A noti-radiolabelled /« .V//M hybridization method for the detection of epidertnal cytokine tnRNA. Exp Dermatol 1992: 1: 230-235. © Munksgaard, 1992
S. R. E. J.
E. M. Home\ D. Aldridge', E. McVittie", Thornton\ E. Ramage^ and A. A. Hunter'
Departments of 'Dermatology and ^Pathology, University of Edinburgh, Edinburgh, Scotland
Key words: cytokine mRNA - FITC-labelled oligonucleotide probes
Abstract: We describe a non-radiolabelled method lor the iti .situ detection of epidertnal cytokine mRNA in paraffm sections of skin biopsies frotn sites of nickel contact sensitivity. The method uses Huorescein isothiocyanate-labelled oligonucleotide exon probe cocktails.
Introduction
The need to identify cells within a tnixed cell population containing a nucleic acid of interest led, in the late 196O's, to the development of in situ hybridization (ISH). The technique involves the annealing under stringent conditions of a complementary probe to cellular DNA or RNA. Sensitivity depends upon effective preservation of the target nucleic acids, specificity of the probe, tnaxitnization of hybridization and an efficient detection system. Probe technology has been revolutionized with the production of oligonucleotide probes. Such probes can be tailored to the gene under investigation and made long enough to reduce non-specific interactions yet short enough to penetrate tissues. Furthermore, as avidity can vary between exon sequences, 'cocktails' of probes to different sequences of nucleic acids within the gene of interest can be produced. Recently, exon cocktail oligonucleotide probes to tnessenger RNA sequences have becotne commercially available. Such probes can be labelled at either the 5' or the 3' end depetiding on the enzytne system employed. 230
Dr, S, E, M, Howie, Department ot Pathology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland, Tel, 031 650 2955, Accepted tor publication 24 November 1992
Traditionally, probes have been labelled with radioisotopes, but to shorten detection titne with itnproved resolutioti atid to reduce the health hazard we chose to have the probes labelled with tluorescein isothiocyanate (FITG). The FITC-labelled probes used in this study did not provide sufficient signal for direct visualization by UV tnicroscopy and an itnmunohistochemical detection tnethod was employed. Unlike biotin, FITC gives minitnal background for itnmunohistochetnistry as it does not occur in mammalian tissues. Material and methods
Subjects Five females and 1 tnale fully-informed nickel-sensitive volunteers (age range 24 52 years) each of whotn had a positive patch test (2+ positive; International Cotitact Dertnatitis Research Group scale) (1) within the 2 months preceding the study were recruited. All had 25 mg 5"/» nickel sulphate in white soft paraffin (WSP) applied under 8 tntn Finn chatnbers to three sites on the lower abdotnen which had not previously been affected by eczetna. Patches were retnoved at 3 hours after application and 6 mm punch biopsies taken from the center of
Non-radiolabelled /// situ hybridization
the site after infiltrating the surrounding area with 2 % Lignocaine (Astra). Probes Exon cocktail FITC-labelled oligonucleotide probes, for interleukin-la, interleukin-8, and Actin were purchased frotn British Biotechnology, Cowley, Oxford. Ten-microliter aliquots at 20 ng/|.tl were stored at - 7 0 C in autoclaved disposable plastic Eppendorf tubes until use. Sample preparation T h e biopsies were immediately placed into buffered formalin-saline, and embedded in paraffin at 38 C. Eive-tnicron sections were lloated onto sterile 0.1% diethyl pyrocarbonate (SIGMA, UK) water ( D E P C water; 1 tnl DEPC/liter of deionized glass distilled water, shaken until dissolved and autoclaved) and taken onto 3-aminopropyltriethoxysilane ('Tespa') (Sigma) coated slides. Sections were dried overnight at 56 C and stored in dust-free conditions until used. Sections were dewaxed and rehydrated through clean xylene, and ethanol washes, followed by washing in sterile DEPC water. Sections were incubated in 2X statidard sodium citrate (SSC) with a final rinse in sterile D E P C water. Laboratory preparation for in situ hybridization To preserve RNase-free conditions, solutions were made up in 0.1% DEPC water. All glasswear was heated at 250X for 12 h. All disposable plastics were autoclaved before use. Both glassware and plastics were stored dust-free until use. Gloves were worn at all titnes when handling reagents, glassware or plastics.
Mannheim) in 50 mM Tris-HCl buffer pH 7.6 at 37 C for 60 min enhanced hybridization. The reaction was terminated by washing in phosphate-buffered saline (PBS) and fixing with paraformaldehyde. Preh vbridization, hybridization and posthybridization washes After prelitninary studies exatnining the effects of varying the constituents of the hybridization buffer tnixture (HBM), the cotnposition of HBM which gave the most satisfactory results was established and is shown in Table 1. All reagents were supplied by Sigtna and, where possible, were of molecular reagent grade. Slides bearing single sections were ptehybridized with 60 |il HBM/section and incubated in a humid chamber for 60 tnin at 37 C. Prior to hybridization, HBM was drained off the slide. The labelled probe was then added at various concentrations in HBM. In this study each probe was used at a concentration of 20 ng probe/ 10 pl HBM/section. Gelbond (Flowgen Instrutnents Ltd., England), cut to shape, was placed onto each section. Overnight iticubation at 37 C in a sealed hutnid container was followed by several washes of SSC of decreasing ionic sttength. Probe detection, enhaitcenient and visualization Experiments to detect the probe were undertaken with three monoclonal and one polyclonal antiFITC antibodies. DAKO polyclonal rabbit antiFITC (V403) was the preferred antibody as there was minitnutn background staining. Before use, one part of the antibody was absorbed overnight at 4 C with one part of swine .serutn, one part of whole hutnan blood, and a finely cut preparation of 500 tng whole human skin. The absorbed antibody was then ultracentrifuged and affinity-puri-
Slide preparation To ensure tissue sections retnained on the slides during the prolonged ISH procedure, tnictoscope slides were prepared in the following tnanner. They were soaked in 1% Decon for 1 h, washed in running tap water for a tninitnutn of 30 tnin. After washing in 4 changes of distilled water, they were taken up through 157« ethanol, industrial methylated spirit, 95% ethanol and then air-dried. Each slide was dipped individually in freshly prepared 2 % 'Tespa' in acetone, washed in 2 rinses of DBPC water, air-dried and stored in dust-free conditions. Pretreatnients Prelitninary work showed that incubation of sections with 1 |.ig/ml proteinase K (Boehringer
Table 1. Hybridization Buffer Mixture Constituents
Concentration
Sodium chloride
0.6 M
Sodium pyrophosphafe
0,1%
PVP
0,2%
RGOII
0,2%
EDTA Tris-HCl pH 7,6
5 mM 50 mM
10%
Dextran sulphate DNA (sheared salmon sperm)
10%
'Formamide (deionised)
50%
' Formamide was deionised with mixed bed resin [Sigma M8032] tollowing
the insttucfion sheet
231
Howie et al.
fied on a Protein G column (Phartnacia), in accordance with the tnanufacturer's protocol. Sections were incubated with purified anti-FITC antibody, washed and then, following further incubation with biotinylated anti-rabbit itnmunoglobulins [swine F(ab)n fraction, DAKO E 431], a sensitive streptavidin and biotinylated alkaline phosphatase system was used to detect the hybridization signal [in situ Hybridisation Detection System K 600, DAKO]. This results in an insoluble blue chromogen fortned by the hydrolysis of 5bromo-4-chloto 3 indolyl phosphate (BCIP) and concotnitant enzyme reduction of nitro blue tetrazolium (NBT) at the site of hybridization. Visualization was not improved by counterstaining. Sections were covered with Cristal/tnount (Biogenesis, Bournemouth, England). The final working protocol is given in Table 2. Controls Sections were processed as above with otnission of probe, or with RNase [SIGMA Type 1-A] digestion, 1 mg/ml at 37 C for 60 min prior to prehybridization. Assessment of ISH Microscopic assessment of sections was undertaken by an observer (S.E.M.H.) who was blind to the clinical or the laboratory hybridization procedutes. The effects of many variations on the protocol were examined during development of the tnethod. Results
The protocol given in Table 2 was that which gave the tnost satisfactory results. Figs. 1 and 2 illustrate representative results obtained frotn the 6 nickel sensitive patients included in this study. Fig. 1 shows the hybridization obtained with IL8 probe and shows the effect of pretreatment with RNase illustrating the specificity of the reactions for mRNA. Fig. 2 shows the patterns of hybridization seen in a different patient with probes for IL-l a, IL-8 and actin. Discussion
Messenger RNA has a short life due to the ubiquitous presence of endogenous ribonuclease, an enzytne particularly tesistant to degradation. Thus ISH is an exacting technique requiring an RNasefree environtnent. fn these studies the biopsy was rapidly placed into ]{)'%, bulTered formol saline and 232
Table 2, In Situ Hybridization Protocol Stock Solutions 20XStandard saline citrate (SSC), 3 M NaCl, 0,3 M citric acid 10x Phosphate buffered saline (PBS), 1,3 M NaCl, 70 mM NaHjPO,,, 30 mM NaH^PO, 2H 0 Profeinase K, 1 mg/ml in 50 mM Tris-HCl pH 8,0 containing 1 mM CaCI Stored at 4''C, lOxTris-HCI, 0,5 M, pH 7,6, Tris buffered saline (TBS): 50 mM Tris-HCl, 150 mM NaCl, pH 7,6, Ail procedures are carried out at room temperature, unless otherwise stated uewaxitig and Rehydration 1. Xylene 2. 100% ethanol 3. 95% ethanol 4. 70% efhanol 5, 2XSSC (prewarm to 60°C) 6, DEPC wafer Proteinase K Digestion 1, I X Tris-HCl 2, Proteinase K: 1 ),ig/ml in 1 x Tris-HCl 3, ixPBS 4. 0.4% paratormaldehyde in 1 x PBS 5, DEPC water
2x5 1x1 1x1 1x1
mins min min min
1x10 min at 60°C 1x1 min 1 x 5 mins 1x60 mins at 37 °C 1 x 5 mins 1x20 mins at 4°C 1 x5 mins
RNase Digestion Prepare RNase: 100 mg/ml in 2xSSC, 10 mM MgClj 1, Apply RNase to each section 1 x60 mins at 37 °C 2, 0,4% paraformaldehyde in 1 xPBS 1 x20 mins at 4''C 3, DEPC water 1 x 5 mins 4, Air dry Sections NCT tor RNase treatment: 1, 2XSSC, 10 mM MgCI^ 1 x60 mins at 37 °C Postfix, wash and dry as for RNase digestion Pretiybridization 60 |.il HBM/section
1x60 mins at 37°C
Hybridization Lcnfrol sections: 10 pi HBM/section Test sections: - 2 0 ng probe/10 pl HBM/section Post Hybridization Washes Float oft cover slips in 2xSSC Prewarm SSC solutions at 37 °C 1, 2XSSC 2, 1XSSC 3, 0,5 X SSC 4, 0.2 X SSC 5, TBS with 2 mM MgClj, 0,1% BSA, 0,1% Triton 100
37°C overnight 37 °C overnight
2 x 5 mins 37°C 2 x 5 mins 37°C 2 x 5 mins 37°C 2 x 5 mins 37°C 1x15 mins
Detection Procedure All reagents were diluted in and all washes were carried out in TBS with 0,1% BSA 1, 20% swine serum 1 X10 mins 2, Affinity purified rabbit anfi FITC (1:100) 1x30 mins 3, Rinse each section and wash 3x2 mins 4, Biotinyiafed anti-rabbit Igs (1:500) 1x30 mins 5, Rinse each section and wash 3 x 2 mins 6, 'Streptayidin 1x30 mins 7, Rinse each section and wash 3 x 2 mins 8, "Biotinylated Alkaline Phosphate 1 X30 mins 9, Rinse each section and wash 3x2 mins 10, Distilled water 1 x2 mins 11, *BCIP/NBT substrate 1 x60 mins in dark 12, Distilled water 1 x5 mins 13. Mount in Cristal/mount * From DAKO in situ hybridisation system [K600], as per schedule.
Non-radiolabelled in situ hybridization -»>>**
-C Figure I. In situ liybridiz;ition demonstrating epidermal cytokine mRNA in nickel-challenged skin of a nickel-sensitive subject, (a) no probe, (b) IL-S iiiRNA expression, note the cytoplasmic signal and the absence of nuclear staining, (e) absence of IL-8 niRNA expression rollowing RNa.se digestion. Original magnification x 200.
sections were post-fixed in 0.4'Xi parafortnaldehyde following proteinase K digestion. Both fixatives penetrate tissue rapidly, inactivate RNase and preserve tissue tnorphology. RNA is proteinase-resistant and it has been shown that in ISH, particularly where fixed rather than frozen sections are used, proteinase digestion increases the signal to noise ratio (2). No signal
•%
Fii;i(re 2. Hybridization patterns seen in a further subject, (a) no probe, (b) IL-la mRNA expression, (c) IL-S mRNA expression, aclin niRNA expression. Note the variation of slaining pattern with dilTercnt probes. Original magnification x 100,
233
f
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was detected in our systetn if proteinase K pretreatment of sections was otnitted. This is analogous to the pretreatment of sections with trypsin to reveal certain antigenic moieties in immunohistochemical procedures. However, this enzymatic digestion can increase non-specific adherence of either the probe or the imtnunohistochetnical detection system and thus background staining (3). Tn our studies increasing proteinase K concentration was accompanied by increasing non-specific nuclear staining. Although it is reported that proteinase K increases signals by partially removing the cellular proteins that itnpair probe access (4), it is also possible that it increases tissue valency which, in turn, may increase non-specific probe adherence by electrostatic attraction. Many non-radioisotopic methods have been used to label probes (5), but their sensitivity, particularly in paraffin sections, has been questioned (6). Reeently, a non-radioisotopic ISH technique has been shown to be highly sensitive provided the system is optitnized and sufficiently stringent conditions are employed to tninimize non-specific binding (6). Digoxigenin-labelled probes have been used previously in skin (7), but non-specific adherence has been teported in other tissues (8). In our studies we chose to employ an FITC label as this hapten is not naturally found in tnammalian tissues and thus its detection ought not to be accotnpanied by excessive endogenous background. Initially, amplification was attetnpted by using several antiFITC monoclonal antibodies. The results were disappointing and so we opted to use an anti-FITC polyclonal antibody. Although this resulted in a much enhanced signal it was accompanied by increased non-FITC specific staining. This background was successfully retnoved by absorption of the polyclonal anti-FIT'C with a variety of tissues including skin, followed by affinity purification on protein G. Atnplification of the signal obtained with the anti-FITC antibody was initially attempted with an avidin bridge and detected using Fast Red or Fast Blue as the chromogen for biotinylated alkaline phosphatase but this proved ineffective. Acceptable signal detection was consistently achieved with the more sensitive strepavidin and BCIP/NBT procedure. Messenger RNA degrades with increasing temperature atid alkalinity and it has been foutid that the addition of formamide enables the optimal temperature of annealitig to be reduced. It is also known that the presence of sodium chloride will inlluence annealing by stabilizing the complex. In our studies, satisfactory results were obtained when 50"/) fortnatnide was used with 0.6 M sodium chloride at pH 7.6 at 37 "C overnight. 234
Following hybridization under low-stringency conditions, washing in conditions of high stringency, low salt and high temperature will remove probes with mismatched sequences. In our studies prolonged washing with decreasing ionic coticentrations of SSC tnarkedly reduced background staining. The concentration of the probe used is critical in tnaxitnizing hybtidization. Dextran sulphate is added to the probe mixture; it appears to act as a phase separator as its tnolecular weight prevents it entering the cell and thus effectively raises the concentration of probe in the tissue-diffusible mixture (9). In our studies the addition of dextran sulphate was found to enhatice probe visualization without increasing background. Non-specific binding of probe to cellular tnRNA would be expected to have resulted in identical hybridization patterns regardless of the probe used, as the same amount of DNA was applied to each section. Fig. 2 illustrates that different probes produee quite distinctive staining patterns, indicating that the binding detected is due to differences in the distributions of the mRNAs tested. Positive ISH for cytokine mRNA in the epidermis of nickel-sensitive subjects following antigeti exposure indicates that transcription has occurred but does not confirm the production of protein in significant quantities (translation). Additionally, the technique at ptesent is not atnenable to fortnal quantification. Nevertheless, despite these inherent disadvantages ISH is a powerful tool and unlike Northern blotting allows an individual cell producing specific tnRNA to be identified within tissues. In summary, we report a successful method of detection of cytokine mRNA by non-radiolabelled in situ hybridization. We are currently using this protocol to study iti detail the immediate transcription of cytokine genes in contact sensitivity. The results of these studies will be reported elsewhere.
References 1. Fregert S. Patch testing. In: Manual of Contact Dermatitis. Copenhagen: Munksgaaid, 1981: 71-81. 2. Angerer L M, Angerer R C, Detection of poly A-t-RNA in .sea urchin eggs and embryos by quantitative in situ hybridisation. Nucleic Acids Research 1981: 9: 2819-2840. 3. Curran R C. Gregory J. The unmasking of antigens in paralTm sections of tissues by trypsin. Expetentia 1977: 33: 1400-1401. 4. Naylor M S, Balkwill F R. Northern analysis and //; situ hybridisation of eytokinc mRNA, In: Balkwill F R. ed, Cytokines. Oxford: IRL Oxford Llniveisity Press. 1991: 31 50. 5. Holler H. Principles of in situ hybridisation, ln: Polak J M, McGee J O'D, ed. //; situ Hybridi.sation Principles and Practice. Oxford: Oxford University Press. 1990: 15-29. 6. Fleming K A, Evans M, Rylcy K C, Franklin D, Lovell-
Non-radiolabelled in situ hybridization Badge R H. Morey A L. Optimisation of non-isotopie in .situ hybridisation on formalin-fixed, paraffin-embedded material usine digoxigenin-labelled probes and transgenic tissue J Patholouy 19'92: 167: 9 17, 7 Fisher C. Angus Ii. Rees J. //; siiu hybridisation using digoxigenin labelled probes in human skin, Br.l Derm 1991: 125: 516-520.
8. Mitchell B S, Dhami D. Schumacher U. //; ,s//i( hybridisation: a review of methodologies and applications in the biomedical sciences. Mcd Lab Sci 1992: 49: 107-118, 9. Warlord A. In situ hybridisation: a new tool in pathology. Med Lab Sci 1988:45:381-394,
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