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.whita...@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@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@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 > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.utsouthwestern.edu _mailman_listinfo_histonet&d=DwICAg&c=k9MF1d71ITtkuJx-PdWme51dKbmfPEvxwt8SFE kBfs4&r=BgjUe6oLZB0OAcW6Y6Rn-n0Q03Ac4dWC2x8Sg24AeiY&m=gzHjMQcM0W22Saad2E8Pgv a_UOfvxrD1xD0IQ57lDUY&s=rVkT1YjvFe8uXhvIGPusCI6h-qfQm2I-v86i1XIRePc&e= > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.utsouthwestern.edu _mailman_listinfo_histonet&d=DwICAg&c=k9MF1d71ITtkuJx-PdWme51dKbmfPEvxwt8SFE kBfs4&r=BgjUe6oLZB0OAcW6Y6Rn-n0Q03Ac4dWC2x8Sg24AeiY&m=gzHjMQcM0W22Saad2E8Pgv a_UOfvxrD1xD0IQ57lDUY&s=rVkT1YjvFe8uXhvIGPusCI6h-qfQm2I-v86i1XIRePc&e= _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet