What's wrong with my FFPE FISH?

• 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.

• 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.

• Ensure all paraffin is cleared with extra washes of xylene (or substitute) over a longer time period.

• 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.

• 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.

• 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.

• 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.

• 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.