C.Non-radioactive in situ hybridisation on paraffin embedded sections

From:Dr. Qiling Xu
Division of Developmental Neurobiology,
National Institute for Medical Research, The Ridgeway,
Mill Hill, London NW7 1AA, UK.
E-mail: q-xu @nimr.mrc.ac.uk

Pretreatments and hybridisation
1.Dewax slides in Toluene for 10 min, twice.
2.Rinse with 100% ethanol for 1.5 min, three times.
3.Rehydrate through 70%, 50%, 30% ethanol, each step for 1 min.
4.Immerse in saline for 5 min.
5.Immerse in PBS for 5 min.
6.Rinse in 2 x SSC twice
7.Treat with 10 ug/mi proteinase K in PBS for 10 min. This step will need to be optimised for different tissues.
8.Fix sections in 4% PFA in PBS, 20 min.
9.Rinse in 2X SSC twice
10.Dehydrate through 50%, 75%, 100% methanol, then air-dry.
11.Add hybridisation mix including DIG-labelled probe at 200-400 ng/mi. Cover with coverslip and incubate in a humidified box at 650C.

Posthybridisation washes and detection of probe

1.Incubate slides in 25% formamide, 2X SSC at 65 oC until coverslips fall off
2.Rinse in 2X SSC twice
3.Incubate in 2X SSC at 65 oC 2 times for 30 min each
4.Incubate in 0.2X SSC at 65 oC 3 times for 30 min each
5.Wash in PBT, 2 times for 10 min each.
6.Lay slides flat in a humidified chamber and overlay with blocking solution for 30 min.
7.Replace with blocking solution containing 1/5000 diluted AP-anti-DIG antibody, and incubate for 1 hr.
8.Rinse 2 times and then wash 3 times for 15 min. in TBT.
9.Wash with NTMT 2 times for 10 min.
10.Overlay sections with colour developing solution in a humidified dark chamber, until colour has developed (usually overnight).
*(See below for composition of solutions)

Solutions:

PBS:phosphate buffered saline. Prepare usmg Dulbecco "A" tablets (Oxoid)

PBT:PBS, 0.1% Triton X-100

TBS:50 mM Tris HCl pH 7.5, 150 mM NaCl

TBT:TBS, 0.1% Triton X-l00

TESPA is 3-aminopropylethoxysilane: Sigma number A3648 (toxic, so take precautions)

Proteinase K: 10 mg/mi stock in sterile dH2O

PFA fixative: 4% paraformaldehyde in PBS. Heat at 650C with occasional agitation until dissolved and cool on ice. Use on the day of preparation. Note: take precautions with paraformaldehyde fumes which are toxic.

Glutaraldehyde: 25% stock solution (Sigma)

20X SSC stock solution: 3 M NaCl, 0.3 M sodium citrate, pH 7.0

Hybridisation mix for Method 1: 50% formamide, 5 x SSC, 50 ug/mi yeast RNA, 50 ug/mi heparin, 0.1% Tween-20.

Hybridisation mix for Method 2: 50% formamide, 5 x SSC, 5 x Denhardts solution, 250 ug/mi yeast RNA, 500 ug/mi salmon sperm DNA.

Blocking solution for Method 1: 2% sheep serum, 2 mg/mi BSA in PBT

Blocking solution for Method 2:10% sheep serum in TBS

NTMT:100 mM Tris HCl pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Triton X-100

NBT stock solution: 75 mg/mi NBT (Boehringer) in 70% dimethylformamide

BCIP stock solution: 50 mg/mi BCIP (Boehringer) in dimethylformamide

Colour developing solution: add 4.5 u1 NBT and 3.5 u1 BCIP per mi of NTMT

Equipment and materials

Cryostat (or microtome if sectioning wax-embedded embryos instead)
Glass slides
Slide racks and contalners
Sandwich boxes to use as humid chambers
Oven or water bath at 65oC

For further details of this method, and discussion of the theory and practical aspects of in situ hybridisation, see:

"In situ Hybridisation: A Practical Approach" D.G. Wilkinson, IRL Press, Oxford, 1992.

Nieto, M.A., Patel, K. and Wilkinson, D.G. (1996) In situ hybridisation analysis of chick embryos in whole mount and tissue sections. Meth. Cell Bio. 51, 29-235.