section7
Both the stability and the rate of formation of cRNA probe : target mRNA hybrids are temperature dependent 46. In filter and solution hybridisations, the specificity of the probe is increased and the background reduced at high temperatures. However, tissues become damaged by prolonged exposure to high (> 60oC) temperatures and trap radiolabelled probes in the damaged areas. The effect of temperature of S/N ratio in ISH is summarised below.
The optimum hybridisation temperature is in the range 50oC - 55oC. The exact temperature is probe and tissue dependent
The components of the hybridisation buffer can affect the rate of hybrid formation, the stability of the hybrids formed and may also block the generation of non-specific background. Sulphur-labelled probes interact with free sulphhydryl groups in tissue via the a-thiol group and create a relatively higher background than other radiolabels.
The buffer used in this protocol contains 300 mM NaCl, 10 mM Na2HPO4, 10 mM TrisCl, 5 mM EDTA, 50% formamide, 5% dextran sulphate, 100 mg / ml tRNA and 100 mg / ml degraded herring sperm DNA. 10 mM DTT need only be used for 35S-labelled cRNA.
Dextran sulphate prevents non-specific binding of cRNA 47 and is an essential component of the buffer. It is much more efficient than blocking agents such as PEG 6000. However, low-grade dextran sulphates can generate very poor S/N ratios 48. Other blocking reagents - Blotto, Heparin and Dehardt's solution - will also decrease background when used with cRNA probes, but are they offer no significant advantage over tRNA or ssDNA.
The probe concentration affects both the specific hybridisation signal and the background 49. In the original 1984 paper by Cox et al. 50, the signal continued to increase until the probe concentration reached 2 mg / ml.
With a-32-P, a-33-P and a-35-S-labelled cRNAs, probe concentrations of ~ 200 ng / ml or above increase background labelling, especially with a-35-S. The gain in signal is offset by the concomitant increase in background. However, the low intrinsic backgrounds of 5,6H-labelled RNAs allows them to be used at ~ 1 mg / ml.
Once generated, background appears to be very stable 51. With oligonucleotide probes, high backgrounds resist even washing at high temperatures in water
This general principle also appears to be true for ISH using cRNA probes. Post-hybridisation 'melts' 5 - 10oC above the hybridisation temperature, and digestion with RNase A both improve the S/N, but cannot adequately compensate for high backgrounds
Numerous, often very convincing artefacts are generated during ISH. The use of appropriate controls is essential for subsequent interpretation of the slide autoradiographs.
In addition to the test probe(s), the controls I routinely use are :
¥ antisense probe complementary to a highly expressed mRNA in the tissue of interest
¥ tissue known to express the target mRNA at high levels
¥ sense probe on all types of tissues used
¥ pre-treatment of the tissue sections with RNase A
(¥ X-ray film autoradiography)
These control for :
¥ adequate tissue handling
¥ hybridisation efficiency of test probe(s)
¥ non-specific binding
¥ autoradiographic failure at the liquid film emulsion stage
¥ positive / negative chemography
An ideal positive control tissue expresses the mRNA in only a small subpopulation of cells. The non-expressing areas act as an internal negative control. A specific signal will be seen with the antisense but not with the sense probe. The signal will be reduced or abolished by pre-treatment of the sections with RNaseA.
Use 300 mg / ml RNase A (Sigma, Ribonuclease A, type II-A from bovine pancreas, Catalogue number R5000) for 1 - 2 hours AFTER the proteinase K digestion step but BEFORE paraformaldehyde post-fixation. This ensures that the nuclease is inactivated during hybridisation, and that a decrease in signal is not due to degradation of the cRNA probe. In tissue that has been well-fixed, eg by perfusion-fixation, the signal will not be completely abolished by RNase A digestion, but it should be decreased.
Non-specific binding will be seen with both the sense and antisense cRNAs. These may be binding to other RNAs, in which case the signal will be decreased by RNase digestion. The cRNAs may also bind to non-RNA components. There are a variety of well-documented tissue-specific artefacts and there are individual remedies 52. Some may prove refractory to all treatments.
The labelled probe is resuspended in hybridisation buffer and then hybridised under a coverslip, with the dehydrated tissue sections
NaCl / TrisCl / EDTA Sigma ® pg 40 / pg 27 / pg 27 Na2HPO4 BDH ® pg 18 Formamide Merck, proanalysi, Catalogue number 1.09684 (1 litre) Mixed Bed Resin BioRad, AG 501-X8 resin Dextran sulphate Pharmacia, Catalogue number 17-0340-01 (100g / 500 g) Dithiothreitol Sigma ® pg 39 tRNA Boehringer Mannheim ® pg 40 ssDNA Sigma, degraded herring sperm DNA - crude oligonucleotides, Catalogue number D3159 (10g) Sterile MilliQ water
Hybridisation chamber Large plastic container and stackable trays eg Decor 35 cm x 25 cm x 12 cm deep storage box and Decor plastic trays. These are slightly smaller than the box. There is ~ 1 cm between the bottoms of the trays when stacked Coverslips Menzel Glazer Deckglasser, 22 x 32 mm / 22 x 22 mm / 22 x 40 mm / 22 x 50 mm / 22 x 60 mm, depending on the size of the tissue sections Hybridisation oven 50oC - 60oC
1 10 x hybridisation salts in sterile MilliQ water and autoclave. pH should be < 7.0 and is usually ~ 6.8 ¥ 3 M NaCl ¥ 100 mM Na2HPO4 pH 6.8 ¥ 100 mM TrisCl pH 7.5 ¥ 50 mM EDTA pH 6.8 2 Deionise formamide over mixed bed resin. If the pH is > ~7.4, discard it and try a fresh stock 3 50% (w/v) dextran sulphate in sterile MilliQ water. Store at room temperature 4 10 mg / ml tRNA Dissolve in sterile MilliQ water, extract twice against acid-phenol:chloroform, isopropanol-LiCl precipitate, wash with 70% ethanol, resuspend in sterile MilliQ water. Store in small aliquots at -20oC 5 10 mg / ml ss DNA Dissolve in sterile MilliQ water, extract twice against TE-buffered-phenol:chloroform, isopropanol-Na acetate precipitate, wash with 70% ethanol, resuspend in sterile MilliQ water. Store in small aliquots at -20oC 6 1M DTT for a-35S-labelled probes 7 Air-dry, digested, post-fixed and dehydrated sections 8 Humidify the hybridisation chamber. Place two pieces of Whatmann paper or equivalent in the bottom of the chamber and wet with 1 x hybridisation salts, 50% formamide in sterile MilliQ water. The 35 cm x 25 cm x 12 cm box needs ~ 50 ml
1 Resuspend the probes in hybridisation buffer at : ¥ 100 ng / ml a-32P-labelled cRNA ¥ 50 - 200 ng / ml a-33P-labelled cRNA ¥ 50 - 100 ng / ml a-35S-labelled cRNA ¥ 1 mg / ml 5,6H-labelled cRNA 2 For 1 ml of hybridisation buffer add : ¥ 100 ml 10 x hybridisation salts 1 x ¥ 500 ml deionised formamide 50 % ¥ 100 ml 50% dextran sulphate 5% ¥ 10 ml 10 mg / ml tRNA 100 mg / ml ¥ 10 ml 10 mg / ml ssDNA 100 mg / ml ¥ 10 ml 1 M DTT (a-35S only) 10 mM ¥ x ml probe to concentrations in (1) ¥ y ml sterile MilliQ water to 1 ml 3 Mix the probe and hybridisation buffer thoroughly - it is quite viscous. Apply the following volumes to the centre of the section :
¥ ~ 20 - 25 ml of probe for a 22 x 22 mm coverslip
¥ ~ 30 - 35 ml of probe for a 22 x 32 mm coverslip
¥ ~ 40 - 45 ml of probe for a 22 x 40 mm coverslip
¥ ~ 50 - 55 ml of probe for a 22 x 50 mm coverslip
¥ ~ 60 - 65 ml of probe for a 22 x 60 mm coverslip4 Gently lower the coverslip and catch the surface of the probe-hybridisation buffer mix before letting it go of the coverslip 5 Incubate at least 8 hours at 50oC - 55oC 6 Make up the 1 x hybridisation salts, 50% formamide in sterile MilliQ water wash buffer for the following day and warm to the hybridisation temperature
1 Use deionised formamide to make up the hybridisation buffer 2 Some grades of dextran give very high backgrounds. I have found Pharmacia to give good results as long as it is not frozen. Store the 50% stock at room temperature. As long as it is made up with sterile MilliQ water, it should be OK 3 The coverslips can be used straight from the packet, without further cleaning. Occasional boxes will give a particulate background over the slide. However, coverslips are very fiddly to clean - I don't think it is justified on a routine basis 4 There is no need to seal around the coverslip as long as the volume of probe + hybridisation buffer is that indicated in (3) and the chamber is kept fully humidified 5 Formamide vapour is toxic - use it in a fume hood, especially when hot 6 The probes can be stored at 4oC in hybridisation buffer : ¥ a-32P-labelled cRNA use as soon as possible, due to the short half life of the isotope ¥ a-33P-labelled cRNA can use up to 3 - 4 weeks later, but only if the target is abundant ¥ a-35S-labelled cRNA can use up to 8 weeks later ¥ 5,6H-labelled cRNA use as soon as possible, due to the rapid radiolysis of the probe Probes in water / DTT / RNasin should be stored frozen at -70oC
The coverslips are removed and the sections then washed in a formamide buffer. This removes the free probe, dextran and blocking agents. The slides are rinsed to remove the formamide, and then the non-specifically bound probe is digested with RNase A. Digested probe is removed with two high temperature washes before the sections are washed and dehydrated ready for exposure to X-ray film
10 x hybridisation salts ® pg 50 Formamide ® pg 50 : lower grades can be used at this stage NaCl / TrisCl / EDTA Sigma ® pg 40 / pg 27 / pg 27 RNase A Sigma, Ribonuclease A type I-AS from bovine pancreas, Catalogue number R5503 (100 mg) PBS PBS tablets, Oxoid ® pg 15 Sodium citrate as tri-sodium salt, BDH, molecular biology grade, Catalogue number 43607 (100g / 1 kg) Sterile MilliQ water Alcohols Reagent grade 70%, 95% and absolute alcohols, BDH
Slide racks Plastic slide racks ® pg 23 Trough to fit slide racks Plastic troughs ® pg 23
1 Make up the 1 x hybridisation salts, 50% formamide in sterile MilliQ water wash buffer for the following day and warm to the hybridisation temperature 2 5 x RNase buffer in sterile MilliQ water and autoclave ¥ 3 M NaCl ¥ 100 mM TrisCl pH 7.5 ¥ 50 mM EDTA pH 6.8 3 RNase A, 20 mg / ml in sterile MilliQ water. Store in small aliquots at -20oC ¥ 175 g NaCl 4 Sterile PBS 5 20 x SSC buffer in sterile MilliQ water and autoclave ¥ 175 g NaCl ¥ 88 g tri-sodium citrate ¥ dissolve in 900 ml of MilliQ water ¥ adjust pH to 7.0 with NaOH ¥ make up to 1 litre with MilliQ water
1 Remove coverslips from the sections - fume hood - hot formamide vapour is toxic Pale a beaker of warm 1 x salts - 50% formamide wash buffer in a spill tray. Immerse the slides : the hybridisation buffer is so viscous that the coverslips should dislodge easily. Slide the coverslips rather than pull them off - it damages the tissue less 2 After the coverslips have been removed, transfer the slides to a black plastic rack sitting in ~150 ml of warm 1 x salts - 50% formamide wash buffer in a black plastic trough 3 Wash the slides at the hybridisation temperature with gentle rocking either 1 x salts - 50% formamide 30 minutes 50oC x 6 or 1 x salts - 50% formamide 2 hours 50oC x 2 4 Rinse off the formamide in RNase digestion buffer 1 x RNase digestion buffer 5 minutes 22oC x 3 5 Digest non-specifically bound probe with 20 - 40 mg / ml of RNase A 60 minutes 37oC 6 Rinse with PBS 5 minutes 22oC x 2 7 Wash with 2 x SSC 30 minutes 65oC x 2 8 Rinse with sterile MilliQ water 3 minutes 22oC x 2 9 Dehydrate through graded ethanols : 70% alcohol 3 minutes 22oC 95% alcohol 3 minutes 22oC Absolute alcohol 3 minutes 22oC Absolute alcohol 3 minutes 22oC 10 Air-dry the sections ready for X-ray film autoradiography
1 Do not allow the slides to dry out at any stage during coverslip removal 2 If the first washes are not performed in the 1 x salts - 50% formamide buffer, there is a danger of precipitating the dextran sulphate + probe over the tissue sections 53 3 Dispose of the first two 1 x salts - 50% formamide buffer washes as hot waste 4 The optimal RNase A concentration is determined by both the probe and the tissue 5 Dispose of the RNase A wash and the first PBS rinse as hot waste
46 J. Meinkoth and G. Wahl (1984) Hybridisation of nucleic acids immobilised on solid supports Anal Biochem 138:267-284 47 J. H. Pringle, P. V. Senior and A. Warford (1989) In situ hybridisation to cellular mRNA using single-stranded cRNA probes labelled with 35S In situ hybridisation course. Course manual. Revised 1990 48 D. M. Simmons, J. L. Arriza and L. W. Swanson (1989) A complete protocol for in situ hybridisation of messenger RNAs in brain and other tissues with radio-labelled single-stranded RNA probes J Histotechn 12:169 - 181 49 G. Niedobitek and H. Herbst (1991) Applications of in situ hybridisation International Review of Experimental Pathology. Ed Richter, G.W. Solez, K. 32:1 - 56. Harcourt Brace Jovanovitch 50 K. H. Cox, D. V. DeLeon, L. M. Angerer and R. C. Angerer (1984) Detection of mRNAs in sea urchin embryos by in situ hyrbdisation using asymetric RNA probes Dev Biol 101:425 51 J. D. Penschow, J. Haralambidis, S. Pownal and J. P. Coghlan (1989) The location of gene expression by hybridisation histochemistry using oligodeoxyribonucleotide probes Methods in Neurosciences 1 (Gene Probes):222 - 238 52 G. Niedobitek and H. Herbst (1991) Applications of in situ hybridisation International Review of Experimental Pathology. Ed Richter, G.W. Solez, K. 32:1 - 56. Harcourt Brace Jovanovitch 53 J. H. Pringle, P. V. Senior and A. Warford (1989) In situ hybridisation to cellular mRNA using single-stranded cRNA probes labelled with 35S In situ hybridisation course. Course manual. Revised 1990