178
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
I Subprotocol 6
Detection
Materials
• Wash solution A: 50% formamide (inexpensive grade), 2x SSC, pH 7.0.
• Wash solution B: 1x SSC- 0.1x SSC, p 7.0. The stringency should be adjusted empirically for each probe: e.g. if a probe also hybridizes weakly to
a second region, stringency should be increased. If a probe of a different
species is used, stringency conditions should be decreased.
• Wash solution C: 4x SSC, 0.1% Tween20; pH 7.
• Wash solution D: 2x SSC, 0.05% Tween 20.
• Blocking solution: 3% BSA, 4x SSC, 0.1% Tween 20.
• Detection buffer: 1% BSA, 4x SSC, 0.1% Tween 20.
• Antifade-mounting solution. Such solutions considerably decrease the
bleaching of the illuminated slides. Several commercial reagents are
available; of these V ecta Shield (V ector Laboratories) proved to be
very efficient. Alternatively, DABCO antifadingmedium can be prepared
as follows: 0.233g of DABCO (1,4-Diazabicyclo[2.2.2]octane), 20 mM
Tris-HCl, pH 8.0, 90% glycerol. Store this solution in the dark at 4°C.
Procedure
Detection Note: During the entire procedure, the slides must not be allowed to dry
completely.
1. Prewarm washing solutions A and B in a 42°C and 60°C water bath, respectively.
2. After taking the slides out of the humidified chamber, carefully remove
the ruhher cement using a forceps.
3. Place slides in a Coplin jar containing wash solution A prewarmed to
42°C, agitate in a shaking water bath for 10 minutes until coverslips
fall off. Transfer slides to another jar containing wash solution A, and
agitate for 5 minutes. Change the wash solution A twice more. Shake
for 5 minutes each.
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
I Subprotocol 6
Detection
Materials
• Wash solution A: 50% formamide (inexpensive grade), 2x SSC, pH 7.0.
• Wash solution B: 1x SSC- 0.1x SSC, p 7.0. The stringency should be adjusted empirically for each probe: e.g. if a probe also hybridizes weakly to
a second region, stringency should be increased. If a probe of a different
species is used, stringency conditions should be decreased.
• Wash solution C: 4x SSC, 0.1% Tween20; pH 7.
• Wash solution D: 2x SSC, 0.05% Tween 20.
• Blocking solution: 3% BSA, 4x SSC, 0.1% Tween 20.
• Detection buffer: 1% BSA, 4x SSC, 0.1% Tween 20.
• Antifade-mounting solution. Such solutions considerably decrease the
bleaching of the illuminated slides. Several commercial reagents are
available; of these V ecta Shield (V ector Laboratories) proved to be
very efficient. Alternatively, DABCO antifadingmedium can be prepared
as follows: 0.233g of DABCO (1,4-Diazabicyclo[2.2.2]octane), 20 mM
Tris-HCl, pH 8.0, 90% glycerol. Store this solution in the dark at 4°C.
Procedure
Detection Note: During the entire procedure, the slides must not be allowed to dry
completely.
1. Prewarm washing solutions A and B in a 42°C and 60°C water bath, respectively.
2. After taking the slides out of the humidified chamber, carefully remove
the ruhher cement using a forceps.
3. Place slides in a Coplin jar containing wash solution A prewarmed to
42°C, agitate in a shaking water bath for 10 minutes until coverslips
fall off. Transfer slides to another jar containing wash solution A, and
agitate for 5 minutes. Change the wash solution A twice more. Shake
for 5 minutes each.