[Histonet] FISH question

[Gudrun Lang]

Thanks for your advice.
I use adhesive slides from Leica. Years ago we had Superfrosts of another
brand (maybe Thermo) and I can't remember similar issues.
Can you recommend a special brand for FISH?
Have you ever tried to do FISH on a slide without adhesion?

Gudrun

-----Ursprüngliche Nachricht-----
Von: Whitaker, Bonnie [mailto:Bonnie.Whitaker at osumc.edu] 
Gesendet: Dienstag, 19. Februar 2019 18:54
An: 'Mark Tarango'; Gudrun Lang
Betreff: RE: [Histonet] FISH question

I know a few years ago, we ran into the same issue, and the problem actually
was with the slides.  We were using cheaper slides for most of histology at
the time, but had to purchase "higher end" slides for the FISH.

Thanks,
Bonnie

Bonnie P. Whitaker
AP Operations Director

The Ohio State University
Wexner Medical Center
Department of Pathology
N305 Doan Hall
410 West 10th Avenue
Columbus, Ohio  43210

614.293.8418
FAX 614.293.2779
Pager: 614.293.7243 ext. 5013

-----Original Message-----
From: Mark Tarango via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, February 19, 2019 12:43 PM
To: Gudrun Lang
Cc: HistoNet
Subject: Re: [Histonet] FISH question

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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