[Histonet] grad student problem

[Caroline Miller]

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