LCM and IHC Re: [Histonet] storage of frozen cryosections (again)
I think the key point here is cryostat storage works for LCM protocols.
However, for general IHC without using LCM in the end, we avoid cryostat
storage. Sections taken from cryostat temperature get water condensation
formation on sections which contributes to morphology and antigen damage.
We do not have problems with fresh frozen section morphology but I think
that is more dependent on fixation.. Acetone fixation is notorious for
poor morphology, but if the antigen can be fixed with another fixative, say
acetone/alcohol mixture, one sees a huge improvement in morphology
preservation. Obviously, PFA is even better.
We know in our lab, as do many others, that PFA fixation for our murine
CD4 and CD8 markers leads to no staining of these lymphocytes. I
patiently await the day when we could use PFA for murine CD4 and CD8, alas
- no clones yet nor a retrieval that works.
At 10:08 AM 2/2/2005, you wrote:
>We pick up our sections and put them in a slide box that is in the
>cryostat and keep them there until we are finished sectioning. Then we
>rush them to the -80 freezer. We put a dessicant pack or two in the box
>with the slides, and we wrap the slide box in foil, then stick that into a
>plastic bag that we label as to what the slides are. When we are ready to
>do the next step, we remove the box from the freezer, quickly take out
>the slides we need & put the box back. We then place a warm finger under
>the section for half a second, then put the slide into cold fix or 70%
>alcohol, just depends on what we are using the slides for. It is usually
>LCM for RNA. but it could work for other things like IHC. The morphology
>is terrible on fresh frozen tissue sections, so for IHC we always perfuse
>the animal with PFA and store the tissue in in 20% sucrose, then freeze
>the tissue for cryosections. Then they can air dry because they are fixed.
>Make sense? I hope this helps.
>Kathleen Spencer HT (ASCP)
>Lab Manager/LCM Supervisor
>On Feb 2, 2005, at 9:53 AM, Anna Elisse Beaudin wrote:
>> I have a question regarding the storage and preservation of frozen
>>cryosections. I apologize if I have asked this question before, but
>>I'm still unclear as to the best approach. Currently, when I collect
>>cryosections, I dry them overnight at room temp and than use them
>>immediately the next day. however, I would like to be able to collect
>>sections to use at later timepoints. My concern is that O/N drying at
>>room temperature followed by freezing and storage at -20 or -80 might
>>cause ice crystals or otherwise affect the quality and morphology of my
>>sections. Additionally, even if I were to freeze the sections
>>immediately following sectioning, they are still collected on slides at
>>room temperature, and thus I wonder if I would have the same problem
>>with thaw/freeze. I would greatly appreciate anyone's advice with
>>this... I would really like to be able to preserve more sections for
>>Thanks so much in advance!
>>Division of Nutritional Sciences
>>Histonet mailing list
>Histonet mailing list
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Veterinary Molecular Biology
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