Preserving Endothelial Cells in Frozen 'Large' Blood Vessels Sections

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glycophile
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Preserving Endothelial Cells in Frozen 'Large' Blood Vessels Sections

Hello,
Currently I am doing alot of sectioning of frozen human and mouse aortas and am interested in an endothelial antigen.  My problem is that we seem to be ripping off the endothelial cell layer during sectioning and I am loosing my cells of interest.
We prep these vessels by embedding fresh aortas in OCT and freezing in a dry ice/isopentane slurry.  We are cutting circular cross-sections of these aortas and essentialy are staining aortic rings.  We are sectioning 5-10 um thick sections with a Lieca machine.  In general the morpholgy of the sections is great with no rips or folds.  However ~90% of the endothelial cells are gone after sectioning.  With a DAPI staing, we seen very few endothelial cell-shaped nuclei (flattened, oblong shaped nuclei at the very edge of the tissue).  With vWF or CD-31 staining, we see very little poisitive, lumenal signals, but when we see them their nuclear morphology are the flattened, oblong shaped nuclei I described above.  Sometimes, we can see the endothelial cell layer (CD-31 positive) still barely hanging onto the vessel wall or sometimes we see a completetly disconnected fragment of tissue (CD-31 positive) trapped in the lumen of the section, as if it had ripped off the wall but still stuck to the slide during sectioning/fixation. 
Does anyone have any suggestions about how to preserve the integrity of the endothelial cell layer in aorta cross sections?  Also, we don't see this problem in smaller vessels.  For instance, in an aortic section where the endothelium is gone or barely hanging on, we will get consistent CD-31 positive staining of the Vasa vasorum (blood vessels of the aorta), so we think its something about the shearing forces imparted during sectioning that the aortic endothelial cell layer can't handle.
Thanks so much.
Chris

Jason King
Jason King's picture
Is it possible to put

Is it possible to put something like OCT/sucrose into the aorta before snap freezing. This might help protect the endothelial cells from the freezing process and the sectioning. OCT/sucrose works well in the delicate mouse lung when used to inflate the lung prior to snap freezing in LN2.

ProtocolFinder
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Im with Parvo on this one,

Im with Parvo on this one, can you perfuse with the heart with OCT prior to removing the aorta?
 
Rus

glycophile
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I will give 50/50 OCT/PBS and

I will give 50/50 OCT/PBS and 100% OCT a try. Any suggestions as to the ratio of PCT/Sucrose? 50/50? Danke!
Chris

Jason King
Jason King's picture
I've had a look back in my

I've had a look back in my notes but so far, no luck. I think it was a 1:1 mixture of OCT and 30% Sucrose. I would use OCT/sucrose rather than OCT/PBS.

Jason King
Jason King's picture
I have also heard of people

I have also heard of people perfusing with a fixative, using the natural circulation whilst the mouse is anaethetized (eg. PFA). Would this improve the endothelial structure or just mask the CD31 epitopes?

Arvind Singh Pundir
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glycophile wrote:

glycophile wrote:

Hello,
Currently I am doing alot of sectioning of frozen human and mouse aortas and am interested in an endothelial antigen.  My problem is that we seem to be ripping off the endothelial cell layer during sectioning and I am loosing my cells of interest.
We prep these vessels by embedding fresh aortas in OCT and freezing in a dry ice/isopentane slurry.  We are cutting circular cross-sections of these aortas and essentialy are staining aortic rings.  We are sectioning 5-10 um thick sections with a Lieca machine.  In general the morpholgy of the sections is great with no rips or folds.  However ~90% of the endothelial cells are gone after sectioning.  With a DAPI staing, we seen very few endothelial cell-shaped nuclei (flattened, oblong shaped nuclei at the very edge of the tissue).  With vWF or CD-31 staining, we see very little poisitive, lumenal signals, but when we see them their nuclear morphology are the flattened, oblong shaped nuclei I described above.  Sometimes, we can see the endothelial cell layer (CD-31 positive) still barely hanging onto the vessel wall or sometimes we see a completetly disconnected fragment of tissue (CD-31 positive) trapped in the lumen of the section, as if it had ripped off the wall but still stuck to the slide during sectioning/fixation. 
Does anyone have any suggestions about how to preserve the integrity of the endothelial cell layer in aorta cross sections?  Also, we don't see this problem in smaller vessels.  For instance, in an aortic section where the endothelium is gone or barely hanging on, we will get consistent CD-31 positive staining of the Vasa vasorum (blood vessels of the aorta), so we think its something about the shearing forces imparted during sectioning that the aortic endothelial cell layer can't handle.
Thanks so much.
Chris

are you giving fixation to your tissue at any step to preserve the integrity of the cells, as it seems your pre-embedding  procedure is ok as you had mentioned the morphology looks ok but during further post sectioning procedure may be your tissue is not maintaining the integrity or you are not covering the tissue fully with the embedding media(OCT) at the edges and all

lwang
lwang's picture
You might try the following

You might try the following two approaches:
1. Get a thicker frozen tissue section, such as 20 um, plus OCT perfusion before you snap freeze the tissue;
2. Try  paraffin section. I know some anti-PECAM-1 antibodies work for fixed papffin section.