Pat Heslop-Harrison and Trude Schwarzacher
Update: 1 February, 2006 9:57 AM
Practical In Situ Hybridization
Printer: select text, then file-print-selection

'It doesn't work': first suspect is your water; second your pH meter or technique. Use purified water by RO, ion exchange or distillation; in many cases you might need two approaches, and always suspect central building water supplies. Only after checking water, check you other chemical reagents! Did you use the hydrated or anhydrous salt (e.g. for 1M phosphate buffer, use 14.2g of Ha2HPO4 or 26.8 g of Na2HPO4.7H2O; Tris powder is slightly hygroscopic and hence should be stored in sealed containers). To inhibit growth of microorganisms, we usually use sterile, purified water to make up solutions. Salt and Tris solutions should be autoclaved before storage.

1M Tris-HCl buffer

Many molecular biology protocols use buffers based on Tris (tris[hydroxymethyl]amino-methane; called Trizma by Sigma). This has good buffering capacity in the biologically relevant pH range (around the pKa of Tris, 8.1 at 25C), it does not change hybridization stringency nor inhibit enzymes because it does not contain the elemental cations (Na sodium, K potassium, Mg magnesium) found in many buffers and it does not precipitate Ca, Mn or Ag salts added to it.

It has some unusual properties compared to elemental ion buffers: the pH of Tris solutions changes drastically with temperature, decreasing by 0.3 unit with a 10 °C increase in temperature; it also decreases 0.4 unit with dilution from 1M to 10 mM.; many pH electrodes do not measure pH of Tris correctly. The pH given in protocols here refer to the undiluted stock solution (1 M for Tris) at 20 ° C, with + 0.1 unit accuracy (i.e., the pH is not that of the solution after dilution).

Tris-HCl buffer stock solutions (normally 1M) can be

  1. purchased as sets (e.g. from Sigma);
  2. made by mixtures of from stock solutions of 1M Tris base (121.1 g / l ) and 1M Tris-HCl ('Tris hydrochloride') (157.6 g/ l) (see Table)
    pH at 25C 1M Tris-HCl (ml) 1M Tris base (ml) Tris-HCl g/l for 0.05 M solution Tris-base g/l for 0.05 M solution
    7.2 917.4 82.6 7.02 0.67
    7.4 869.6 130.4 6.61 0.97
    7.8 719.9 280.1 5.32 1.97
    8.0 612.4 387.6 4.44 2.65

    ; or
  3. made by mixing Tris base (121.1 g l -1 ) with 1M HCl. To make a 1M solution, dissolve Tris base (121.1 g) in 900 ml water, add 65 ml (pH 7.4), 55 ml (pH 7.6) or 35 ml (pH 8.0) 1 M HCl, adjust pH with an additional 5 to 8 ml of 1M HCl, and make up to 1 l with water.
Some electrodes do not measure the pH of Tris solutions accurately, and electrodes specified for Tris should be used. Solutions made by mixing Tris base and Tris-HCl will be accurate enough to use with pH measurement. Tris-HCl buffer should be sterilized by autoclaving.

500mM EDTA pH 8
Dissolve 186.1 g disodium ethylene diamine tetraacetate.2H 2 O in 800 ml of water. Add about 10 g NaOH pellets, stir on a magnetic stirrer and adjust to pH 8 with further NaOH (about 10 g pellets). Make up to 1 l and sterilize by autoclaving

10x TE-buffer (Tris-EDTA buffer)

Mix 100 mM Tris-HCl buffer of the required pH with 10mM EDTA pH 8

10x NTE-buffer (NaCl-Tris-EDTA buffer) also called STE-buffer (Sodium-Tris-EDTA buffer)

Mix 5M NaCl, 100mM Tris-HCl pH7.5, 10mM EDTA pH 8

20x SSC (saline sodium citrate, salt sodium citrate or standard saline citrate)

This is a widely used, weak buffer which is used for many washes and to control stringency during in situ hybridization. The 20x stock solution consists of 3M sodium chloride and 300mM trisodium citrate. 20x SSC is used for two reasons: it is the strongest that readily goes into solution, while fungi and bacteria do not grow readily in this strength. To make the stock, dissolve 175.3 g NaCl and 88.2 g Na 3 C 6 H 5 O 7 .2H 2 O in 900 ml water. Adjust to pH 7 with NaOH or HCl if necessary, make up to 1 l and sterilize by autoclaving.

Phosphate buffers
Phosphate buffers (normally sodium phosphate, although potassium or mixtures are used regularly) are made by mixing disodium phosphate and monopotassium phosphate of the given molarity in water to reach the required pH. Phosphate buffer can be kept for a short time at 4°C. 1M stock solutions: A: dissolve 14.2g Na 2 HPO 4 (or 26.8g Na 2 HPO 4 . 7 H 2 O) in 100ml water (final volume) B: dissolve 13.6g KH 2 PO 4 in 100ml water (final volume) As examples to make the working buffer: 100 mM at pH 6.9: dilute A and B to 100 mM and mix together. For pH 6.9, 60ml of 100mM Na 2 HPO 4 + 40ml of 100mM KH 2 PO 4 . 6mM at pH 7.4: Dilute stock solutions and mix 6mM Na 2 HPO 4 and 6mM NaH 2 PO 4 to get pH7.4. 75mM at pH 7: 61.5ml of 75mM Na 2 HPO 4 + 38.5mM of 75mM NaH 2 PO 4 (Protocol 6.3)

10x PBS (Phosphate buffered saline)

PBS is widely used in cell culture and for washing or treatments of preparations, and contains 120 mM saline in phosphate buffer. Stock solutions are usually made at 10x and diluted to 1x before use. PBS can be bought ready mixed as concentrate or powder for dilution (e.g. from Sigma). Alternatively make a 10x stock solution, autoclave and keep at 4°C and dilute before use. Many formulations include 2.7 mM KCl, but this is not necessary in the applications in this book; the NaCl concentation can be 138 mM or 145 mM in other formulations. To make 10x PBS (1.3 M NaCl, 70 mM Na 2 HPO 4, 30mM NaH 2 PO 4 ), add 76.0 g NaCl, 12.46 g Na 2 HPO 4 .2H 2 O and 4.68 g NaH 2 PO 4 .2H 2 O to 900 ml water, adjust pH to 7.4 with 1 M NaOH or HCl, and make up to 1 l with water before autoclaving.

1x TBS (Tris buffered saline)

TBS is recommended as an alternative to PBS or SSC in some applications. Make up 50mM Tris-HCl with 150mM sodium chloride and adjust pH to 7.6 with hydrochloric acid.

1x Detection buffer

Detection buffer is used for rinsing slides after detection, with a detergent to help uniform spreading and rinsing of previous solutions, and a high salt concentration to avoid removing labels. It consists of 4x SSC (diluted from 20x SSC) with 0.2% (v/v) Tween 20, but PBS or other buffers can also be used