Tips include how to optimise tissue enzyme digestion results, why you should avoid baking or aging of slides, and advice on improving cell morphology.
When using the enzyme solution to digest your FFPE slide, liberally apply the enzyme ensuring the section is covered, as evaporation may cause the enzyme to recede from the edges and cause inconsistent digestion.
Jenny Morse
FISH Field Application Scientist, North America
If your cells appear intact with a three-dimensional look to them and there is visible cytoplasm using a phase contrast microscope you could try re-fixing your cells in 1:1 methanol:acetic acid.
Ashley Hart
FISH Field Application Scientist, North America
Try pre-fixing your bone marrow or peripheral blood samples by slowly adding ice-cold fix with agitation immediately after the hypotonic treatment to improve your cell preparations.
Graham Halford
FISH Field Application Specialist, Europe
Consider using a mild hypotonic treatment before fixing CD138+ selected cells, which may improve cell morphology.
Laurence Cambridge
FISH Field Application Specialist, Europe
Positively charged slides should only be used for FFPE sections. If these slides are used for cell suspensions, they may produce high green background.
Ashley Hart
FISH Field Application Scientist, North America
After pre-treatment, consider using DAPI to assess for over/under digestion of FFPE sections; this can be washed off before applying the FISH probe.
Jenny Morse
FISH Field Application Scientist, North America
When using archived cell pellets, consider re-fixing using fresh fixative to ensure they provide optimal results.
Laurence Cambridge
FISH Field Application Specialist, Europe
Consider using Pepsin to digest excess cytoplasm and cellular debris in Cytospin samples to improve hybridisation quality.
Graham Halford
FISH Field Application Specialist, Europe
Try cleaning your slides in 70% ethanol before use, to get rid of any dust or debris. A high degree of debris may lead to a high background.
Jenny Morse
FISH Field Application Scientist, North America
Try to avoid baking or aging of slides (if not needed for another application e.g. G-Banding) as it may reduce signal fluorescence.
Laurence Cambridge
FISH Field Application Specialist, Europe
Perform tissue enzyme digestion at 37°C on a hotplate for best results.
Graham Halford
FISH Field Application Specialist, Europe
When performing enzyme digestion, why not try using Parafilm® to cover the area. This may aid spreading of the reagents and reduces enzyme evaporation.
Ashley Hart
FISH Field Application Scientist, North America
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