[Histonet] FISH question

[Gudrun Lang]

Dear histonetters!

I have difficulties with my FISH preparation on FFPET. I struggle with
massive background. It looks like a thick fluorescent film.

The signals can't be seen because of the background. Even the nuclei are
hard to see.

The background is within the tissue but also surrounds it. Therefore it must
be directly on the glass slide.

 

The slide is clear after deparaffination and after pretreatment with citric
buffer and pepsin. After the pepsin the slides are rinsed in 2xSSC, then
50%-70%-96%-100% ethanol (p.a.).

And then the slides are airdried. 

On the dry slides foggy streams appear. The slides become turbid. When I
rinse them again in graded ethanols it becomes better but still a little
turbid.

After hybridisation and stringent washing the slides are air-dried again and
coverslipped with Dapi. 

When looking at the slides in the fluorescence microscope the trouble
arises. 

 

My assumption is, that there is a remnant of the salt of the SSC buffer. How
can I inhibit this deposit? Can I replace the buffer with water without any
harm to the tissue?

Or ist there a different cause for the turbidiy? 

I use fresh reagenses from xylene to buffer and ethanol. 

Any hints are welcome.

 

Thanks in advance 

Gudrun Lang