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