[Histonet] FISH question

[Mark Tarango]

Hi Gudrun,

Are you sure you have digested long enough with pepsin?  If the tissue is
not well digested you will see background.  We use sodium thiocyanate for
pretreatment reagent, not citric buffer.  These are my first thoughts.

Mark

On Tue, Feb 19, 2019 at 9:18 AM Gudrun Lang via Histonet <
histonet at lists.utsouthwestern.edu> wrote:

> 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
>
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