In situ hybridization

In this section:

Nucleotide Stocks

5x SP6 Buffer

MEMFA

PTw

Triethanolamine 0.1 M

PTw Containing 4% Paraformaldehyde

Hybridization Buffer

SSC, 20X

Denhardt’s solution, 100X

RNase A

RNase T1

4% BMB Blocking Reagent in MAB

Alkaline Phosphatase Buffer

Levamisol 1 M

NBT/BCIP

PVA Alkaline Phosphatase Buffer

Bouin’s Fixative

Bleaching solution (Mayor et al. 1995)

Nucleotide Stocks

2.5 mM nucleotide mix with digoxigenin-11 UTP (Boehringer Mannheim 1209 256; 25 moles)

10 mM CTP - 10 ul

10 mM GTP - 10 ul

10 mM ATP - 10 ul

10 mM UTP - 6.5 ul

10 mM dig-11UTP - 3.5 ul

Alternatively, prepare the whole tube of dig-UTP (25 moles; Boehringer Mannheim 1209 256) at one time to made a 2.5 mM stock.

100 mM CTP - 7.1 ul

100 mM GTP - 7.1 ul

7100 mM ATP - 1 ul

100 mM UTP 4.6 ul

10 mM dig-UTP 25

DEPC-treated water 234 ul

dNTPs 10 mM

dNTPs (Roche), 100 mM (250 ul each)

Mix 250 µl each dNTP = 1000µl Add 1500µl ddH20 for final 2500µl of 10 mM solution.

Make 25-50 ul aliquots in eppendorf tubes. Store at -20C.

5x SP6 Buffer

200 mM Tris (pH 7.5)

30 mM MgCl2

20 mM spermidine

Autoclave and store frozen in aliquots.

MEMFA (MOPS/EGTA/Magnesium Sulfate/Formaldehyde Buffer)

	1X	10X	for 1 L 10X
MOPS (pH 7.4)	0.1 M	1.0 M	209.27 g
EGTA	2 mM	20 mM	7.608 g
MgSO4	1 mM	10 mM	2.46 g
Formaldehyde	3.7%

• Prepare a 10 X solution without the formaldehyde for storage. Add fresh formaldehyde(1/10 volume of standard 30% stock) prior to use. The solution will turn yellow with age and when autoclaved.

PTw

1X PBS(phosphate buffered saline)

0.1% Tween-20

Triethanolamine 0.1 M (pH7.0-8.0; Sigma T 1502)

For 1 L: Dissolve 18.6 g triethanolamine in DEPC water, adjust pH with 8 pellets NaOH. Check pH with drops on pH paper. Adjust volume, store at RT. Do not expose the solution to a pH meter of dubious cleanliness.

PTw Containing 4% Paraformaldehyde

1 volume of 20% paraformaldehyde

4 volumes of PTw

Note: To made 20% paraformaldehyde in water, neutralize the cloudy solution with NaOH and then heat at 65 deg C, with shaking, until clear, Store at -20 dec C.

Hybridization Buffer

50% formamide, redistilled (BRL 5515UB)

5x SSC

1 mg/ml Torula RNA (Type IX, Sigma R 3629)

100 ug/ml heparin (Sigma H 3125)

1x Denfardt's solution

0.1% Tween 20 (Sigma P 1379)

0.1% CHAPS )Sigma C 3023)

10 mM EDTA

Note: Prepare Torula RNA in DPC-treated water. Store in 1 ml aliquots at -20 deg C. It is normal for this solution to be brown and thus it needs no further processing.

For 4 L bottle:

2 L Formamide

1 L 20x SSC

4 g Torula RNA

0.4 g Heparin

40 ml Denhardt’s solution

4 ml Tween 20

4 g CHAPS

Dissolve in dH2O, adjust volume. Store at -20C.

SSC, 20X stock solution

175.3 g NaCl

88.2 g sodium citrate

Dissolve in 800 ml of water, adjust pH to 7.0 with NaOH, and adjust volume to 1 liter.

For 4 L bottle:

NaCl 701.2 g

Sodium Citrate 352.8 g

Dissolve in 3.3 L dH2O, adjust pH 7.0, and volume. Store at RT.

Denhardt’s solution, 100X

2% BSA (ICN 810661)

2% polyvinylpyrrolidone (PVP-40 Sigma)

2% Ficoll 400 (Pharmacia)

For 100 ml 100X:

BSA 2 g

PVP-40 2 g

Ficoll 2 g

Add a small amount of water to ingredients, make a slurry, then dilute. Store at -20C.

RNase A (Sigma R 5000)

Dissolve 10mg/ml in TE (pH 7.8) and boil for 10 min before use. Store aliquots at -20C.

RNase T1(Sigma R8251)

Dissolve 10,000 units/ ml (1000X) in 0.1 M sodium acetate at pH 5.5.

Boil for 10 min before use.

Store aliquots at -20C. While in use keep at 4C, avoid repeated thawing and freezing.

Maleic Acid Buffer (MAB), 10X

100 mM maleic acid (Sigma M 0357)

150 mM NaCl (pH 7.5)

This solution is used for numerous washes so make up liter quantities.

For 4 L bottle:

Maleic acid 464.4 g

350.64 NaCl

Start dissolving by adding around 280-320 g or more NaOH pellets. Add more pellets during the day as they dissolve. Procedure takes all day. Adjust pH to 7.5. Adjust volume. Store at RT.

4% BMB Blocking Reagent in MAB

Dissolve 10g BMB in 100 ml 1X MAB or 50g BMB (the whole bottle) in 500 ml 1X MAB(see above for MAB).

Make 50 ml aliquots in Falcon tubes. Store at -20C.

Alkaline Phosphatase Buffer

100 mM Tris (pH 9.5)

50 mM MgCl2

100 mM NaCl

0.1% Tween 20 (Sigma P 1379)

2 mM levamisol (Sigma)

For 1 L:

100 ml 1 M Tris pH 9.5

50 ml 1 M MgCl2

25 ml 4 M NaCl

1 ml Tween-20

2 ml 1 M Levamisol

Add fresh levamisol just before use. This reagent inhibits endogenous phosphatase. Alkaline phosphatase buffer can be stored, but magnesium hydroxide tens to precipitate over time. A slight misty precipitate has no effect on the reaction.

Levamisol 1 M

Dissolve Levamisol (204.292 g/L) in dH2O, adjust volume, store at -20C.

NBT/BCIP

Nitro blue tetrazolium (NBT): 75 mg/ml in 70% dimethylformamide

5-bromo-4-chloro--3-indolyl-phosphate (BCIP): 50 mg/ml in 100% dimethyl-formamide

PVA Alkaline Phosphatase Buffer

Alkaline posphatase buffer (see above) containing 10% polyvinyl alcohol (Aldrich 36,313-8)

Dissolve PVA (98-99% hydrolyzed, average m.w. 31,000-50,000) to a final concentration of 10% alkaline phosphatase buffer. Heat the solution to help dissolve the PVA. PVA tends to precipitate during storage, so do not store this before for more than a few days. The final buffer is quite viscous, and this the NBT, BCIP, and levamisole must be mixed thoroughly before adding it to embryos. The embryos must also be washed well after staining, to prevent precipitation of PVA in methanol.

Bouin’s Fixative

To make up to 100 ml of Bouin's fix, dissolve 1 g of picric acid in 70 ml of water and then add 25 ml of 37% formaldehyde and 5 ml of glacial acetic acid.

For 50 ml: 35 ml Piric acid 12.5 ml Formaldehyde 2.5 ml glacial acetic acid Adjust volume, store at RT.

Bleaching solution (Mayor et al. 1995)

1% H202 5% formamide 0.5 X SSC(standard saline citrate)