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

• Increase the enzyme digestion time
• Perform the step using a 37°C hotplate
• Ensure that the enzyme is stored at the correct temperature

• Calibrate hotplate/hybridizer 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 hybridizer?

Hotplate pros

• Immediate co-denaturation
• Better temperature regulation

Hotplate cons

• Not ideal for high throughput processing

Hybridizer pros

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

Hybridizer 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 hybridization

• Calibrate hotplate/hybridizer 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 hybridizer?

Hotplate pros

• Immediate co-denaturation
• Better temperature regulation

Hotplate cons

• Not ideal for high throughput processing

Hybridizer pros

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

Hybridizer 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 hybridization