Background Texture
  • Key
  • Problem
  • Identifier
  • Solution
Identifier
  • FFPE sections tear very easily and become difficult to manipulate. Sections 4-6μm thickness are optimal.
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.
  • Store slides in an oven at 50C overnight or 60C for 1 hour to adhere the section to the slide.
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 of xylene (or substitute) over a longer time period.
Excess paraffin Image
Identifier
  • High level of autofluorescence and background.
  • Weak probe signals.
Solution
  • Store both pre-treatment solution and enzyme at 2-8°C.
  • Maintain temperature of water bath at 98-100°C for at least 30 minutes.
  • Treat with enzyme at 37°C.
  • Refresh pre-treatment solution after max. 20 slides.
  • Use ceramic jars instead of glass to maintain the temperature.
  • Use plastic balls on the surface of the water bath to reduce evaporation.
Insufficient pre-treatment 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 minutes
  • Lung: 15-20 minutes
  • Lymph node: 10-40 minutes
  • Kidney: 20-25 minutes
  • Colon: 20 minutes
  • Brain: 15-18 minutes

Optimal pretreatment and digestion times vary, please contact a member of the support team for guidance.

 

*These timings are based on independent validation carried out by Cytocell Ltd on a representative set of tissues using the CytoCell Tissue Pretreatment Kit (LPS 100) and should be used for guidance purposes only. This product is provided under an agreement between Life Technologies Corporation and Cytocell Ltd and is available for use in life science research or human diagnostics 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 at the correct temperature.

 

Suggested digestion times:*

  • Breast: 10-40 minutes
  • Lung: 15-20 minutes
  • Lymph node: 10-40 minutes
  • Kidney: 20-25 minutes
  • Colon: 20 minutes
  • Brain: 15-18 minutes

Optimal pretreatment and digestion times vary, please contact a member of the support team for guidance.

 

*These timings are based on independent validation carried out by Cytocell Ltd on a representative set of tissues using the CytoCell Tissue Pretreatment Kit (LPS 100) and should be used for guidance purposes only. This product is provided under an agreement between Life Technologies Corporation and Cytocell Ltd and is available for use in life science research or human diagnostics only.

Under digestion Image
Identifier
  • Tissue fragmentation.
  • Degraded probe.
  • High background.
Solution
  • Calibrate hotplate/hybridiser to ensure that the correct denaturation temperature is achieved.
  • 75°C for 5 mins is recommended; however increasing the denaturation temperature up to 85°C may improve signal strength in difficult specimens.

 

Hotplate or hybridiser?

 

Hotplate pros

  • Immediate co-denaturation.
  • Better temperature regulation.

Hotplate cons

  • Not ideal for high throughput processing.

 

Hybridiser pros

  • Easy-to-use, less manual input.
  • High throughput.

Hybridiser cons

  • Temperature inconsistencies across positions.
  • Loss of humidity can lead to signal drop out; ensure that the humidity strips are pre-soaked in a 37°C water bath prior to hybridisation.
Over denaturation Image
Identifier
  • Absent or very weak probe signals.
Solution
  • Calibrate hotplate/hybridiser to ensure that the correct denaturation temperature is achieved.
  • 75°C for 5 mins is recommended; however increasing the denaturation temperature up to 85°C may improve signal strength in difficult specimens.

 

Hotplate or hybridiser?

 

Hotplate pros

  • Immediate co-denaturation.
  • Better temperature regulation.

Hotplate cons

  • Not ideal for high throughput processing.

 

Hybridiser pros

  • Easy-to-use, less manual input.
  • High throughput.

Hybridiser cons

  • Temperature inconsistencies across positions.
  • Loss of humidity can lead to signal drop out; ensure that the humidity strips are pre-soaked in a 37°C water bath prior to hybridisation.
Under denaturation Image
FISH tips & troubleshooting

All laboratories should validate their own protocol and analysis guidelines according to their regional guidelines. This webpage is for general FFPE FISH troubleshooting, following standard cytogenetic practices such as those found in the AGT Cytogenetics Laboratory Manual (2017), and is not suggesting intended uses for any CytoCell product nor should any be inferred by the reader.

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