STRINGENT WASHES For further details of washing stringency and Excel spreadsheet for calculation with SSC and formamide concentrations and temperatures, see In situ stringency calculations http://www.le.ac.uk/biology/phh4/string.htm

 

REAGENTS
  1. 2x and 0.1x SSC: dilute from 20x SSC stock .
  2. Formamide: good but not the highest grade. Immediately after purchase, store at 20 °C in aliquots sufficient for the washes in one experiment, typically 40 ml (Note 1). Formamide is a potential carcinogen
  3. Stringent wash solution: typically, use a final concentration of 20% formamide and 0.1x SSC for high stringency (40 ml 100% formamide and 1 ml 20x SSC made up with water to 200 ml) or 2x SSC (40 ml 100% formamide and 20 ml 20x SSC made up to 200 ml) for low stringency (Note 2)

 

METHODS

For the washing steps, slides are put into staining jars and covered totally with solution. For steps 4 to 7 use a gentle shaking water bath, rotating platform or hand-agitate the solution. To change solutions, pour liquid off carefully and replace slowly with next solution, avoiding turbulence, or transfer slides to another staining jar with the next solution. Do not let slides dry out between changes of solutions
  • Prepare post-hybridization washing solutions. Typically, for 8 slides in a 100ml staining jar, you will need 500ml 2x SSC, 200ml 0.1x SSC, and 200ml stringent wash solution (the stringent wash solution should be prepared in a fume hood). Heat solutions to 42-45 ° C in a water bath .
  • Take slides from hybridization chamber, avoiding condensation dropping onto the slides. Examine slides and note anything unusual: Have any slides dried out? Has any water dropped on the slides? Are there any bubbles?
  • Float off coverslips by incubating slides in a staining jar of 2x SSC at 35-42 ° C. The plastic coverslips will fall away in the solution and are removed with a pair of forceps. Any surface film or dust must be wiped from the liquid in the jar with a tissue before pouring away solution. Otherwise it will stick strongly to preparations.
  • Wash with fresh 2x SSC at 42 ° C for 2 min
  • Incubate slides in stringent wash solution two times for 5 min at 42 ° C (or temperature as required, Note 2). Measure the temperature in the solution in the staining jar, and remove from water bath or heat to maintain temperature to within 0.5 ° C (Note 3).
  • Wash slides in 0.1x SSC (high stringency) or 2x SSC (low stringency) twice for 5 min at 42 ° C (Note 4).
  • Wash slides with 2x SSC twice for 3 min each at 42 ° C.
  • Take the staining jar containing slides out of the water bath or incubator and leave to cool for 5-15 min.
  • Proceed to counterstaining and mounting (for fluorochrome labeled probes, Protocol 9.3), or antibody detection (Protocol 9.2) or enzymatic detection (Protocol 10.6) for other labels (Fig. 9.1).

 

NOTES

  1. Molecular biology grade formamide will have a pH around 7 and be solid at 20 °C; it degrades at room temperature. Very high quality formamide can be purchased frozen, but for the protocols here, freezing immediately on receipt is adequate if fresh from the supplier. (Sigma catalogue number F7508 is suitable). Formerly, an ion exchange resin (e.g. Amberlite, Merck) was added to purify crude formamide grades not specified for molecular biology.
  2. Adjust stringency as required. See Table 7.2 and Chapter 7 on stringency
  3. This step controls the stringency of hybridization, must include at least one change of solutions, and must be accurate (±0.5 °C).
  4. Formamide needs to be washed away completely; inadequate washing is a source of background. Make sure that slides are covered totally with solution, otherwise dirt will accumulate at the top of the slide and run down to the preparation when changing solution or taking slides out.
  5. Based on Schwarzacher et al. (1994)