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2.6.1 Synthesis of DIG-labelled RNA robe for ISH

2.6.1.1 Restriction enzyme digest; preparation of DNA template

To 5µg DNA extracted from a maxiprep of the appropriate plasmid (2.8.5.4), 5 µl of the appropriate restriction enzyme (Table 2-2), 5 µl of buffer and RNAse free water to a final volume of 30 µl were added and incubated at 37 °C for 2 hours. DNA was then extracted using Phenol:Chloroform:Isoamyl alcohol (25:24:1 [Sigma]), Chloroform:Isoamyl alcohol (24:1[Sigma]) and ethanol, resuspended in 30µl RNAse-free water and stored at -20 °C.

Gene Vector Enzyme used to create sense probe; buffer Enzyme used to create anti-sense probe; buffer Source MHV-68 tRNA; pEH1.4

pBluescript _{buffer (Biolabs)} EcoRI; EcoR1 HindIII; Buffer B _(Roche)

Prof. Stacey Efstathiou CCSP pBluescript XhoI; Buffer H

(Roche)

EcoRI; EcoRI

buffer (Biolabs) Prof. James Stewart SPLUNC1 pBluescript EcoRV; Buffer B _(Roche) SmaI; Buffer 4 _(Biolabs)*

AG2 _SPORT6 pCMV- _{buffer (Biolabs)} EcoRI; EcoRI _{buffer (Biolabs)} XhoI; EcoRI

Dr. Colin Bingle AG3 _SPORT6 pCMV- _{buffer (Biolabs)} EcoRI; EcoRI _{buffer (Biolabs)} XhoI; EcoRI

Table 2-2 Restriction digest enzymes and buffers used to create DNA templates for RNA probes.

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2.6.1.2 In vitro RNA transcription

A DIG RNA labelling kit (Roche) was used to create digoxigenin-labelled RNA probes. 1 µg of purified template DNA was placed in an RNase-free reaction vial and RNase-free water added to make a total volume of 13 µl. This vial was placed on ice and the following reagents added: 2 µl 10xNTP labelling mixture, 2 µl 10x transcription buffer, 1 µl protector RNase inhibitor and 2 µl RNA polymerase. The specific RNA polymerase varied; all anti-sense probes were generated with T7 polymerase; sense probes were generated using T3 polymerase, except in the case of AG2 and AG3, when SP6 polymerase was used. The vials were incubated for 2 hours at 37 °C. To each vial, 2 µl DNase I was added to remove the template DNA and incubated at 37 °C for 15 minutes and then 2 µl 0.2M EDTA (pH 8.0) was added to stop the reaction. To the DNase digested probes, 1 µl of 1 mg/ml yeast tRNA was added followed by ethanol precipitation and resuspension in 45 µl RNase-free water.

2.6.1.3 Alkaline hydrolysis

The optimum length of an RNA probe is between 300 and 500 bp; probes longer than 500 bp may lead to non specific binding during in situ hybridisation

(Brown, 1998) and so longer probes were shortened using alkaline hydrolysis. To the probe, 5 µl 0.4 M NaHCO3 0.6M Na2CO3 (pH 10.2) was added and

incubated at 60°C for a period of time calculated thus: T = Li – Lf

K x Li x Lf

where T = time/minutes, Li = initial length of fragment, Lf = final length of

fragment and K = 0.11 kb/min. Following incubation, the reaction was neutralised with 5 µl of 3 M sodium acetate (pH 4.6, Sigma) followed by ethanol precipitation. The pellet was dried and resuspended in 50 µl RNase- free water.

2.6.1.4 Dot blot analysis

Serial ten-fold dilutions of the DIG-labelled RNA probes in 10 µg/ml yeast tRNA were prepared and 5 µl volumes spotted onto an Hybond N+ membrane (Amersham Biosciences). Using a UV cross-linker the DNA was fixed onto the membrane. The membrane was washed in washing buffer (0.1 M maleic acid,

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0.15 M NaCl [pH 7.5], 0.3 % (v/v) Tween 20) for two minutes, on a shaking platform at RT. It was then incubated in blocking solution (10x Blocking solution [Sigma] diluted in Maleic acid buffer [0.1 M maleic acid, 0.15 M NaCl, pH 7.5]) for 30 minutes, with shaking, as before. This was followed by incubation with antibody solution (anti-digoxigenin-AP FAb fragments [Roche] diluted 1:5000 in blocking solution) for 30 minutes, with shaking. The membrane was then washed twice with washing buffer for 15 minutes each time and then equilibrated in detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5) for 3 minutes. One 4-nitro blue tetrazolium chloride (NBT)/5-bromo-4- chloro-3-indolyl-phosphate (BCIP) tablet (Roche) was dissolved in 10 ml double distilled water (final concentration 0.4 mg/ml NBT, 0.19 mg/ml BCIP, 100 mM Tris buffer pH 9.5, 50 mM MgSO4) and used to incubate the

membrane in the dark. The colour reaction occurred over the following 1 - 3 hours at which point the reaction was stopped by rinsing the membrane in TE buffer for 5 minutes.

2.6.2 In situ hybridisation (ISH)

2.6.2.1 Preparation of slides for ISH

RNA-ISH was performed on 4 % PFA fixed, paraffin wax embedded 5 µm tissue sections on Biobond (British Biocell International) coated slides. Slides were deparaffinised by washing in xylene twice for 5 minutes, in isopropanol twice for 5 minutes, followed by 5 minute washes in 96 % alcohol, 70 % alcohol and distilled DEPC-treated water. Slides were then transferred into coplin jars and washed with PBS for 5 minutes. Proteolysis was performed as follows; 20 minutes incubation with 0.2 M HCl, two 30 minute incubations in 2xSSC with 5 mM EDTA at 50 °C and digestion in proteinase K (1 µg/ml [Roche] in 20 mM Tris [pH 8.0] with 2 mM CaCl2) for 15 minutes at 37°C.

Slides were then washed in 0.2 % (v/v) glycine-PBS for 5 minutes and post- fixed for 4 minutes in 4 % PFA, washed twice for 1 minute in PBS and 15 minutes in PBS with 5 mM MgCl2. Acetylation was the performed using 0.25 %

(v/v) in 0.1 M triethanolamine (pH 7.5, VWR International) for 10 minutes and washed in PBS, twice for 1 minutes and then once for 15 minutes.

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2.6.2.2 Prehybridisation and Hybridisation

Slides were prehybridised for 1 hour at 52 °C in pre-hybridisation buffer (0.1 mg/ml single stranded salmon sperm DNA (Sigma), 0.25 mg/ml yeast tRNA (Sigma), 6 x SSC, 45 % (v/v) deionised formamide (Qbiogene), 5 x Denhardt’s solution (Qbiogene).Slides were removed from the coplinjar and placed in a humidity chamber and tissue sections covered with hybridization buffer (60 % [v/v] deionised formamide, 30 mM EDTA [pH 8.0], 30 mM piperazin-N,N’bis(2- ethanesulfate-acid) [PIPES, pH 7.0, Sigma], 0.9 M NaCl, 6x Denhardt’s solution, 0.01 % [v/v] Triton X-100, 0.2 mg/ml yeast tRNA, 0.25 mg/ml single stranded salmon sperm DNA, 8000 U heparin [Sigma], 62.5 mg/ml dextransulfate solution) containing DIG-labelled probes at varying concentrations (1:100 to 1:500), depending on optimal staining results. Slides were covered with hydrophobic gel-bond film (Combrex Life Sciences) and sealed with rubber glue (Fix-O-Gum), then incubated at 37 °C for 15–18 hours. Following incubation, the gel-bond film was removed and slides returned to a coplin jar for post-hybridisation washing: two 15 minutes washes in 6x SSC with 45 % (v/v) formamide at 42 °C, two 5 minutes washes in 2x SSC at RT and two 15 minute washes in 0.2x SSC at 50°C.

2.6.2.3 Detection of hybridised probes

Prior to detection of the hybridised probes, slides were washed in buffer 1 (100 mM Tris, 100 mM NaCl, pH 7.5) for 1 minute, then 30 minutes in buffer 1 with 2 % (v/v) sterile neutral sheep serum (Sigma) and 0.3 % (v/v) Triton X-100, both at RT. Detection was performed with alkaline phosphatase-coupledanti- digoxigenin antibody (anti-digoxigenin-AP FAb fragments, 1:200, Roche) in buffer 1 with 1 % (v/v) neutral sheep serum and 3 % (v/v) Triton X-100 in a humidity chamber at RT for 2 hours. Slides were washed twice for 15 minutes in buffer 1, then once for 2 minutes in buffer 3 (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5). Detection of hybridised probes used 0.1875mg/ml 4-nitro

blue tetrazolium chloride (NBT; VWR), 0.1 mg/ml 5-bromo-4-chloro-3-indolyl- phosphate(BCIP; Sigma) and 0.05 % (w/v) levamisole (Sigma) in buffer 3 at RT, in the dark, for between 2 and 16 hours. This reaction was stopped by a 10 minute wash in buffer 4 (10 mM Tris, 1 mM EDTA), also in the dark. Slides were finally washed in distilled water and then counterstained 10 seconds with

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Papanicolaou's haematoxylin before mounting with glycer-gel (Dako) and coverslipping. They were left to dry in the dark.