Probe signal - Regional loss of DNA
intensity
- The hybridization
varies within
mixture was unevenly
the tissue
distributed on the
section
160
HARALD RIEDER et al.
Problem
Dim or no
signal
Signals are
speckled
over the
whole slide
Presumed Cause
- The tissue was poorly
digested (the target is
not sufficiently
accessible)
- The probe concentration was too low
- The hybridization
conditions were poor
- Loss of DNA (may be
indicated by weak DAPI
staining)
- The stringency
conditions were too low
Solution
- Prepare new protease solution directly
before use
- Check temperature of the water bath
(exactly 37+/-1 0c)
Check pH of the protease buffer
(exactly 2.0)
Ensure the correct amount of probe.
If working with dual color FISH, only
increase the concentration of the probe
which shows weaker signals.
Check probe hybridization time
(at least 14-16 h)
Check temperature of the hybridization
incubator (37+/-1 DC)
Increase fIXation time
- Do not score the slide if DAPI staining
is weak, choose new section
- Score the slide ifDAPI staining is strong
- Ensure that there are no air bubbles in
the hybridization mixture during
incubation
- Check the concentration of the wash
buffers
- Check temperature of the washing buffer
(73+/-1°C)
- Gently agitate the Coplin jar containing
the slides to improve the washing
- Increase the washing time (5 min)
References
Hopman AHN, van Hooren E, van de Kaa ChA, Vooijs PGP, Ramaekers FCS (1991).
Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Modern Path. 4, 503-513
Hopman AHN, Classen S, Speel EM (1997). Multi-color brightfield in situ hybridization on tissue sections. Histochemistry Cell Biology. 108,291-298
Hyytinen E, Visakorpi T, Kallioniemi A, Kallioniemi OP, Isola 11 (1994). Improved
technique for analysis of formalin fIXed, paraffin embedded tumors by fluorescence
in situ hybridization. Cytometry 16: 93-9
Kopf I, Hanson C, Delle U, Verbiene I, Weimark A (1996). A rapid and simplified
technique for analysis of archival formalin fixed, paraffin embedded tissue by fluorescence in situ hybridization (FISH). Anticancer-Res. 16: 2533-6
Marben K, Mehraein Y, Jensen K, Rehder H (1997). FISH-derived diagnostic criteria for
aneuploidies in paraffin embedded tissue sections. Medizinische Genetik 1/1997.
intensity
- The hybridization
varies within
mixture was unevenly
the tissue
distributed on the
section
160
HARALD RIEDER et al.
Problem
Dim or no
signal
Signals are
speckled
over the
whole slide
Presumed Cause
- The tissue was poorly
digested (the target is
not sufficiently
accessible)
- The probe concentration was too low
- The hybridization
conditions were poor
- Loss of DNA (may be
indicated by weak DAPI
staining)
- The stringency
conditions were too low
Solution
- Prepare new protease solution directly
before use
- Check temperature of the water bath
(exactly 37+/-1 0c)
Check pH of the protease buffer
(exactly 2.0)
Ensure the correct amount of probe.
If working with dual color FISH, only
increase the concentration of the probe
which shows weaker signals.
Check probe hybridization time
(at least 14-16 h)
Check temperature of the hybridization
incubator (37+/-1 DC)
Increase fIXation time
- Do not score the slide if DAPI staining
is weak, choose new section
- Score the slide ifDAPI staining is strong
- Ensure that there are no air bubbles in
the hybridization mixture during
incubation
- Check the concentration of the wash
buffers
- Check temperature of the washing buffer
(73+/-1°C)
- Gently agitate the Coplin jar containing
the slides to improve the washing
- Increase the washing time (5 min)
References
Hopman AHN, van Hooren E, van de Kaa ChA, Vooijs PGP, Ramaekers FCS (1991).
Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Modern Path. 4, 503-513
Hopman AHN, Classen S, Speel EM (1997). Multi-color brightfield in situ hybridization on tissue sections. Histochemistry Cell Biology. 108,291-298
Hyytinen E, Visakorpi T, Kallioniemi A, Kallioniemi OP, Isola 11 (1994). Improved
technique for analysis of formalin fIXed, paraffin embedded tumors by fluorescence
in situ hybridization. Cytometry 16: 93-9
Kopf I, Hanson C, Delle U, Verbiene I, Weimark A (1996). A rapid and simplified
technique for analysis of archival formalin fixed, paraffin embedded tissue by fluorescence in situ hybridization (FISH). Anticancer-Res. 16: 2533-6
Marben K, Mehraein Y, Jensen K, Rehder H (1997). FISH-derived diagnostic criteria for
aneuploidies in paraffin embedded tissue sections. Medizinische Genetik 1/1997.