Having Probelems?
Complication:
DIFFUSE PROBE SIGNALS
This may be related to high-homology regions or
inconsistent cell lines.
Solution:
If diffuse signals are on a cell line, run probe on normal sample to rule out a cell line mutation.
Complication:
NO SPECIFIC SIGNALS & HAZY
Underdigested tissue samples can prevent probe
from entering nuclei.
Solution:
Increase digestion time or protease concentration to eliminate extracellular tissue. Cells should appear dark gray and distinguishable under a light microscope.
Complication:
HIGH LEVELS OF BACKGROUND
Non-stringent washes can cause background
fluorescence in cells and make signal detection
difficult.
Solution:
Add additional wash steps, or repeat post hybridization washes. If issue persists, decrease hybridization time.
Complication:
SMALL, WEAK SIGNALS
Small and faint signals can result from improper
sample preparation, incorrect filter specifications,
or non-optimal hybridization conditions.
Solution:
Ensure fixative was prepared properly and filter specifications are compatible with Empire Genomics’ probes. Time and temperature of hybridization may need to be adjusted as well.
Complication:
CELLULAR DEBRIS & BACKGROUND
If samples have not been properly fixed, inner nuclei
staining or cellular debris can remain in the sample.
Solution:
Make sure to use fresh samples and fixative. Both reagents and glassware should be changed regularly.