Background Texture
  • Key
  • Problem
  • Identifier
  • Solution
Identifier
  • FFPE sections tear very easily and become difficult to manipulate.
Solution
  • Place tissue block on ice for 20 mins before sectioning to enable smooth cutting.
  • Regularly replace your cutting blade.
  • Longer, continuous ribbons are easier to manipulate.
Microtome sectioning Image
Identifier
  • Paraffin wax begins to melt.
  • Sections difficult to mount on the slide.
  • Tissue does not adhere.
Solution
  • Maintain water temperature of 40°C - higher temperatures melt the paraffin wax.
  • Use scalpels/tweezers to manipulate the sections.
  • Ensure good adhesion using positively charged slides.
Slide mounting Image
Identifier
  • Excess paraffin will preferentially bind to FITC.
  • Insufficient paraffin clearing affects the probe’s ability to penetrate tissue.
Solution
  • Ensure all paraffin is cleared with extra washes over a longer time period.
Excess paraffin Image
Identifier
  • High level of autofluorescence and background.
  • Weak probe signals.
Solution
  • Store both solution and enzyme at 2-8°C.
  • Maintain temperature of water bath at 98-100°C for at least 15 minutes.
  • Replace pretreatment solution after each batch (max of 20 slides per wash).
  • Use ceramic jars instead of glass to maintain the temperature.
  • Use plastic balls on the surface of the water bath to reduce evaporation.
Insufficient pretreatment  Image
Identifier
  • Auto-fluorescence.
  • Doughnut or ‘ghost’ nuclei.
  • Absence of DAPI staining in some areas.
  • Destroyed tissue morphology.
  • Loss of target gene signal in some cells.
Solution
  • Decrease the enzyme digestion time.
  • After digestion, stain with DAPI and check under microscope.
  • The number of over-digested cells should be <15% of the total.

 

Suggested digestion times:*

  • Breast: 10-40 mins
  • Lung: 15-20 mins
  • Ovary: 10-20 mins
  • Kidney: 20-25 mins
  • Colon: 20 mins
  • Brain: 15-18 mins

The optimal enzyme digestion time will depend on the age of the block, tissue composition and quality of fixation. Our technical support team are able to offer further help and guidance on optimizing digestion times for your tissue.

 

*These timings are based on independent validation carried out by CytoCell on a representative set of tissues using the CytoCell Tissue Pretreatment Kit (LPS100) and should be used for guidance purposes only.

Over digestion Image
Identifier
  • A cloudy haze across the cells.
  • Inconsistent DAPI staining.
  • Reduced probe signal strength.
Solution
  • Increase the enzyme digestion time.
  • Perform the step using a 37°C hotplate.
  • Ensure that the enzyme is stored correctly.

 

Suggested digestion times:*

  • Breast: 10-40 mins
  • Lung: 15-20 mins
  • Ovary: 10-20 mins
  • Kidney: 20-25 mins
  • Colon: 20 mins
  • Brain: 15-18 mins

The optimal enzyme digestion time will depend on the age of the block, tissue composition and quality of fixation. Our technical support team are able to offer further help and guidance on optimizing digestion times for your tissue.

 

*These timings are based on independent validation carried out by CytoCell on a representative set of tissues using the CytoCell Tissue Pretreatment Kit (LPS100) and should be used for guidance purposes only.

Under digestion Image
Identifier
  • Tissue fragmentation.
  • Degraded probe.
  • High background.
Solution
  • Calibrate hotplate/hybridizer to ensure that the correct denaturation temperature is achieved according to your laboratory guidelines.
Over denaturation Image
Identifier
  • Absent or very weak probe signals.
Solution
  • Calibrate hotplate/hybridizer to ensure that the correct denaturation temperature is achieved according to your laboratory guidelines.
Under denaturation Image
FISH tips & troubleshooting
CTA Icon

Have a FISH question? Our support team is on hand to help, get in touch