[Histonet] grad student problem

[Loralei Dewe]

Completely agree...it sounds like the tissue got freeze dried and I doubt
if any staining you might get after lots of work will be completely
reliable.

Loralei



On Jan 3, 2017 11:02 AM, "Caroline Miller via Histonet" <
histonet at lists.utsouthwestern.edu> wrote:

> I hate to say it but I think these tissues are toast. It seems they had bad
> fixation or handling to start with, and I would question whether the
> tissues were left out to dry before fixation or freezing. Or whether they
> got fixed and then sucrose infiltrated (which is my method of choice if the
> antibody allows)
>
> Also - what type of tissues? If it is lymph nodes then maybe there is too
> much fatty tissue and that is why it is not sectioning.
>
> I have had a hard time with T-cell markers in paraffin so I do see the
> hesitation in paraffin embedding. If you do go that route then certainly
> thaw in formalin, not straight water.
>
> Not much help, sorry! But sometimes it is just better to go back and plan
> the experiment properly in the first place than trying to mess around with
> old bits of tissue - because even if you could get it on the slide, would
> you really trust the results of the immunostaining???
>
> Happy New Year All!
>
> yours,
> mills
>
> On Tue, Jan 3, 2017 at 10:05 AM, Roberta Horner via Histonet <
> histonet at lists.utsouthwestern.edu> wrote:
>
> > I got the following from a grad student here at Penn State. I am not sure
> > how to solve his problem if possible. Does anyone have any suggestions I
> > can forward?
> > Roberta Horner
> > Animal Diagnostic Lab
> > Penn State University
> >
> > "I am having some difficulties sectioning mouse tumor samples for
> > immunofluorescent analysis.  We originally went the OCT route because we
> > are staining for T cell markers and were worried that the heating that
> > occurs during paraffin embedding would compromise the T cell receptor.
> The
> > samples are a little old, but we are hoping to section and stain for
> immune
> > cell infiltrates.  When sectioning with the cryostat, the tissue and OCT
> is
> > quite brittle and the sample is not intact enough to transfer to a slide.
> > Two colleagues have given the following suggestions:
> > 1. Thaw the OCT blocks, remove the tissue, and place in a new block with
> > fresh OCT media.  I've tried this technique on a few practice samples and
> > the new OCT media seems to be less brittle and I'm able to get tissue on
> a
> > slide, however the tissue itself still seems to be poor with either
> freeze
> > or sectioning artifacts.
> > 2. Thaw the OCT blocks in water, remove the tissue and place in formalin
> > overnight, place in 70% EtOH, then paraffin embed.  Section on a
> > microtome.  Check the fluorescently labelled antibody data sheet to see
> if
> > paraffin embedding interferes with binding.  Try to stain and see what
> > happens.
> >
> > I was hesitant to try the second suggestion because I have found no
> > protocol that takes tissue originally stored in OCT blocks and
> subsequently
> > redirects them to formalin and paraffin for microtome sectioning.  If you
> > have any recommendations on how to move forward and section these
> difficult
> > samples, or know anyone at the diagnostics lab or at Penn State that
> could
> > help, that would be much appreciated!"
> >
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>
>
>
> --
> Caroline Miller (mills)
> Director of Histology
> 3Scan.com
> 415 2187297
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