I struggle with high background in my FISH preparation on FFPET slides. I use adhesive slides and it looks like any fluorescenting substance sticks to the surface.
Has anyone seen such artefacts and can provide a reason for it?
I use fresh reagenses.
Protocol shortened:
- Deparaffination (Xylen to dest. water)
- Heat Pretreatment in citric buffer
- Pepsin
- Rinsing in 2x SSC, water, dehydration via graded ethanols, then air dry
- Denaturation and Hybridization (Cell Signal Probe)
- stringent wash in 2xSSC with NP40
- rinse in water, air dry, coverslip with Dapi
I would try the following troubleshooting that might help:
1. Try to spindown the flourecenct antibody/dye for 2 minutes in 10,000 rpm centrefuge before use. This operation will precipitate non-specific remnants particle that has the tendency to stick to slides and increase noise in the background.
2. I would recommend the Fischer slides which I found very clean as compared to other brands I used.
3. Try to mount coverslip on a new slide (without tissue) using the mounting media you have. If you see flourecenct background under the microscope i would use a different mounting media instead.
Hope this will help
Thank you for the reply. In the meantime I switched to silanized slides, prolonged rinsing after citric buffer, put in a rinsing step in water after 2xSSC and more.
It seems, that my problem with the massive, maybe cristallin, deposit and background is fixed. Additionally I test different variants of stringent washing (temp., reagens). It is a hard step by step procedure, but I think I will make it.
Sound great. Switching to different slide is a good idea , as well as the prolonged rinses and washes. The calibration steps of fluorescent staining is indeed time consuming but I'm glad to hear that you are on the good direction. Good luck!
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