Nuclear/DNA staining with frozen sections (OCT embedded)

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douglas.linn
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Nuclear/DNA staining with frozen sections (OCT embedded)

Post by douglas.linn » Mon Jan 22, 2018 7:00 pm

I'm just curious what others are using to broadly stain nuclei/DNA when working with OCT-embedded frozen samples. Most standard IHC/IF protocols utilize acetone or methanol, but these seem to destroy quality of nuclear proteins for IMC, so 191/193 Ir and Histone-H3 (clone D1H2) don't stain well (whereas they are very clean when working with FFPE samples). I don't recall ever seeing a detriment to DAPI staining when using these as fixatives for IF. PFA fixation as alternative seems to slightly improve nuclear integrity but doesn't fix cell surface marker epitopes as well. Any recommendations?
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votti
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Re: Nuclear/DNA staining with frozen sections (OCT embedded)

Post by votti » Thu Jan 25, 2018 7:33 pm

Hi there,

Unfortunately I cannot provide much advice: I fix my in vitro tissues with PFA before cutting and never had problems with Iridum. Also I remember seeing a quite good correlation with IMC Ir and IF Dapi/Hoechst.

In general I recommend to allways use Hoechst after the overnight IMC staining for both OCT and FFPE. It just takes 5 min and in case of any problems it is easy to quickly check a stained section in IF to exclude that bad tissue quality /fixation issues are the culprit for problems with the IMC signal. In my experience Hoechst also remains fluorscent after drying down the tissues.

I am curious to hear if anybody else has experience with OCT and IMC and can comment about this issue!
ssivajothi
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Joined: Thu Jan 11, 2018 9:00 pm

Re: Nuclear/DNA staining with frozen sections (OCT embedded)

Post by ssivajothi » Tue Feb 27, 2018 9:03 pm

Hi Douglas,

I just ran a titration/time point experiment on frozen sections (OCT embedded) with the intercalator.

Results indicate that a concentration 0.3uM to 0.5uM Ir for 5 minutes gives the best nuclear staining comparable to staining on FFPE. More time meant more background/smearing. Concentration was okay up to 1.25uM.
This was on mouse bladder tissue. I will test these conditions on different tissues eventually but this should serve as a good starting point. Hope this helps.

Best,
Santhosh
douglas.linn
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Joined: Mon Jan 22, 2018 4:20 pm

Re: Nuclear/DNA staining with frozen sections (OCT embedded)

Post by douglas.linn » Fri May 25, 2018 6:10 pm

Santhosh,
Thanks for the suggestions. I've tried a few concentrations and incubation timings like you suggested but to no avail in either acetone or methanol fixed cryosections. For PFA-fixed cryosections the 191/193 works totally fine (perhaps that is what you were using?), but in my experience doesn't give as good membrane signal as acetone or methanol
-Doug
ssivajothi
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Joined: Thu Jan 11, 2018 9:00 pm

Re: Nuclear/DNA staining with frozen sections (OCT embedded)

Post by ssivajothi » Wed Jun 20, 2018 2:25 pm

Hi Douglas,

Sorry for the late reply. yes, my tissues were PFA fixed. Is there a reason you need to have them acetone or methanol fixed? Do you get better epitope recognition that way?

Also have you tried the solution Cytof antibodies (clones) on your frozen sections? How do they work?

I'd be interested to hear about your experience.

Best,
Santhosh
douglas.linn
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Joined: Mon Jan 22, 2018 4:20 pm

Re: Nuclear/DNA staining with frozen sections (OCT embedded)

Post by douglas.linn » Wed Jun 20, 2018 2:42 pm

Yes, in my hands acetone or methanol fixation gives better resolution of surface markers than PFA, for example for CD8a clone 53-6.7. PFA fixation is still acceptable and may be fine for downstream image analysis. I have found that 70-80% of antibodies ( whether human and mouse-reactive) that we routinely use for suspension CyTOF work for frozen sections
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