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Chapter 4 | Troubleshooting 25
Diffused Signals
Table 4.2 Troubleshooting diffused signals
Probable Cause
Recommended Action
Tissue dries up during processing
Keep tissue section moist starting from the pretreatment boiling step by:
 Adding respective reagents immediately after decanting solution from
slides.
 Limiting tissue exposure to air for too long before adding hybridization
reagents.
 Adding all working reagents onto the slides before moving them to the
40 °C humidified incubator.
Insufficient washing in 1X PBS
Make sure tissues are washed in 1X PBS twice after protease digestion and
twice again after subsequent fixing in 4% formaldehyde.
Fast Red substrate not freshly
prepared
Prepare Fast Red substrate immediately before use.
Slides are not dried before
mounting
Ensure that slide sections are completely dry before mounting (about 20
min).
Mounting solution contained
alcohol
Use DAKO Ultramount or Innovex Advantage mounting media to mount
your tissue. Avoid any mounting solution containing alcohol.
Poor Cell Morphology
Table 4.3 Troubleshooting poor cell morphology
Probable Cause
Recommended Action
Incorrect pretreatment conditions
See Optimization Experimental Design Layout on page 29.
Sample preparation not fixed
properly
Make sure that freshly-dissected tissues are fixed in 10% NBF or 4% PFA for
16-24 hours.
Section thickness not optimal
Make sure tissues are sectioned at 5 ± 1 μm thick.
Tissue Detachment from Slide
Table 4.4 Troubleshooting tissue detachment from slides
Probable Cause
Recommended Action
Insufficient baking of slides


Verify that 30 min baking step was done.
It may be necessary to increase baking time to 1 hr.
Incorrect pretreatment conditions
Perform full assay optimization procedure to determine optimal boiling time
and protease digestion time.
Improper fixation, reagents, or
concentrations
Make sure the correct concentration of NBF was used to fix the slides in the
respective steps.
Temperature of pretreatment
condition too high
Make sure the temperature is within the tolerance range of 95-100 °C. For
fatty, soft tissue such as breast, adjust to 95 °C.
Protease treatment is too long or
at too high concentration
Reduce protease concentration and/or incubation time.