I think you should try to find out about the cellulose contents of the fern you
are working with, and compare it to that of Arabidopsis. If the fern
(Ceratopteris) contains more cellulose, I think this is the cause of your
problem.
If that is the case I think you should try to use a paraffin wax of higher
melting point (63-65ºC) to assure an infiltration more compatible with the
cellulose. Also make sure that the infiltration is optimal.
Finally if you can use thinner sections (7 µm) that could help also.
René J.
--- On Tue, 8/9/11, Cordle, Angela R angela-cor...@uiowa.edu wrote:
From: Cordle, Angela R angela-cor...@uiowa.edu
Subject: [Histonet] Fern tissue loss during in situ hybridization
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
Date: Tuesday, August 9, 2011, 4:46 PM
Dear Histonet members,
I’m having problems with tissue loss during in situ hybridization (ISH) with
fern tissue. The fern in question is Ceratopteris richardii, and the tissue
(tissue does not contain spores) has been fixed in FAA, dehydrated and embedded
in Paraplast plus. Sections are 7 um, and mounted on Probe on Plus slides. I’m
not an expert at this process, but I’ve done a fair amount of embedding,
sectioning, and staining of various tissues (on Haupt’s coated slies), and I’ve
had success with Arabidpsis ISH. I’ll outline the relevant and troubleshooting
procedures that I’ve gone through here:
1. Varied the fixation times and methods. Nothing seems to make a difference in
the retention of the tissue on the slides.
2. Tried a brand new box of Superfrost Plus slides (the ProbeOn Plus slides are
kind-of old), but the superfrost slides actually seemed to provide worse tissue
retention than the ProbeOn Plus slides. Arabidopsis tissue prepared in parallel
was fine on both slides.
3. For mounting, I float tissue on 37°C DEPC H2O in a waterbath that is free
from lotions, or any other substance that I am aware of that can cause problems
with tissue adherence. I remove the slides with the newly adhered sections
completely vertically. Again, Arabidopsis sections mounted in parallel do not
fall off slides in subsequent procedures.
4. I’ve paid a (perhaps) compulsive amount of time making sure that there are
no blebs or bubbles of water underneath sections after mounting. Additionally,
slides are dried for 1-2 hours (vertically) before baking them at 48°C. I have
found that baking the slides for 2 days, rather than 1, slightly increases the
tissue retention for the fern, but not enough for quality in situ results.
5. Varied the time and temperature of the proteinase K treatment, but the
tissue seems to be falling off the slides before this step anyway. It is gone
after this step for sure, regardless of the time or temperature.
5. Taken a great amount of care to ensure that there is no residual tert butyl
alcohol (we dehydrate through an ethanol series them into 100% TBA before
transitioning to Paraplast) in the paraplast before embedding and sectioning.
Arabidopsis tissue embedded in the same blocks has performed perfectly fine in
ISH.
Okay. So, here are my specific questions:
Could it be that this fern tissue is not sufficiently negatively charged that
the tissue will not adhere to any polyL lysine coated slide?
What the beep should I try next?
In advance, thank you all so much for taking the time to help me out with this
frustrating problem! --Angie
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