12 Chromosome Analysis by Fluorescence in situ Hybridization
181
Subprotocol 8
Signal Amplification
Hybridization signals may be weak especially when small probes are used.
In these cases, amplification of the signal can facilitate the evalution.
Whereas the signal intensity is increased considerably, amplification
also results in a marked decrease in the signal-to-noise ratio. Therefore amplification is a useful procedure only in slides with low background signals.
The following technique using avidin mediated detection was first described by Pinkelet al. (Pinkelet al. 1986). The method is based on the binding of antiborlies directed against the reporter binding molecule (e.g. avidin). These antiborlies carry reporter groups which can be detected byfluorochrome coupled secondary molecules. Subprotocol 8 is designed for the
amplification of biotin-labeled probes.
Materials
• Wash solution C, wash solution D, detection buffer, antifade mounting
solution as in Subprotocol 6.
• Biotinylated anti-avidin antibodies.
Procedure
1. Carefully remove the coverslip from the slide.
2. Transferslidesinto wash solution C and incubate 3 times for 10 minutes
at 42°C.
3. Remove slides from the Coplin jar, drain excess fluid, add 200 J..Ll of detection buffer containing 1-5 J..Lg/ml biotinylated anti-avidin antibody,
cover with a 22x40 mm coverslip and incubate in a humidified chamber
at 37°C for 30 minutes.
4. Wash the slides 3 times for 5 minutes in a Coplin jar containing wash
solution C at 42°C.
5. Remove slides from the Coplin jar, drain and add 200 J..Ll of detection
buffer containing 5 J..Lg/ml avidin conjugated fluorochrome.
6. Proceed with washing steps and chromosome counterstaining as described in Subprotocol 6.
Signal
amplification
181
Subprotocol 8
Signal Amplification
Hybridization signals may be weak especially when small probes are used.
In these cases, amplification of the signal can facilitate the evalution.
Whereas the signal intensity is increased considerably, amplification
also results in a marked decrease in the signal-to-noise ratio. Therefore amplification is a useful procedure only in slides with low background signals.
The following technique using avidin mediated detection was first described by Pinkelet al. (Pinkelet al. 1986). The method is based on the binding of antiborlies directed against the reporter binding molecule (e.g. avidin). These antiborlies carry reporter groups which can be detected byfluorochrome coupled secondary molecules. Subprotocol 8 is designed for the
amplification of biotin-labeled probes.
Materials
• Wash solution C, wash solution D, detection buffer, antifade mounting
solution as in Subprotocol 6.
• Biotinylated anti-avidin antibodies.
Procedure
1. Carefully remove the coverslip from the slide.
2. Transferslidesinto wash solution C and incubate 3 times for 10 minutes
at 42°C.
3. Remove slides from the Coplin jar, drain excess fluid, add 200 J..Ll of detection buffer containing 1-5 J..Lg/ml biotinylated anti-avidin antibody,
cover with a 22x40 mm coverslip and incubate in a humidified chamber
at 37°C for 30 minutes.
4. Wash the slides 3 times for 5 minutes in a Coplin jar containing wash
solution C at 42°C.
5. Remove slides from the Coplin jar, drain and add 200 J..Ll of detection
buffer containing 5 J..Lg/ml avidin conjugated fluorochrome.
6. Proceed with washing steps and chromosome counterstaining as described in Subprotocol 6.
Signal
amplification