Hi Nicola, analyzing your EM graphs, I can recognize at least one methodical problem - a soft or not enough cured resin medium. Several small or even  larger holes are clearly visble in your sections. Some of those small ones in empty areas of sections (lumen of aorta) indicate some water or solvent contamination of the resin. Big holes in the location of the "lamina" show lower resistance of the resin against electron beam forces (softness again).  On the other hand, the elastic tissues (laminas) in the wall of any elastic artery, that make layers between smooth muscle cells always tend to shrink during fixation and dehydration of this specimen resulting in several detachments between layers of diffrent homogenity.  In order to minimize these amost inavoidable artifacts, try to do a slow, longer timed penetration of your specimen followed by enough curing of theresin in the owen. Do not microwave accelerate. Try to use Araldite resins instead, if available.

As far as I see the structure of the endothelial basal lamina in some of your sections, the b.l. is still at it's place, and the detachments happen within the subendothelial connective tissue. This is the lower resistace layer prone to the folding and subsequent detachments.

Hopefully this hepls.  Dimitri

There is a problem is fixation and preservation of the tissue.

There si also teh fact that elastin is there among the basal lamina with has also other componeents (collagen iv, proteoglycans) and those aggregate with teh basal lamina which is onlya  tiny layer under the endothelium, with its lamina rara.

You need to improve the fixation...Look at Inoue and Leblond paper from Anatomical Record awhile ago about this structure preservation and discussion, plus the fact this is a blood vessels with elastin, that is not discussed in that paper...

Good luck

Maybe change support for grid collection or have small mesh without that plastic over the grid, so you avoid bubbles

Hi Nicola, I can recognize at least one methodical problem though analyzing your EM microphotographs. You need to improve fixation method. It is best that the mouse was transcardially perfused with PBS (0.1 M, pH 7.4) and then fixed with 200 ml of 0.5% glutaraldehyde, 4% formaldehyde and 15% saturated picric acid in phosphated-buffer (PB, pH 7.4). Then blocks containg aortic rings were removed and postfixated in above fixation for 2h.

Hopefully this hepls you.

Jinlian

Thank you all for the input. It's much appreciated. Some of you suggest that fixation should be improved, which was my hunch too, since during that particular dissection it took longer than expected to take the artery out, cut it into rings and put in the fixative. Jin-Lian Li, I was also considering doing perfusion fixation next time, how long would you do that for before cutting rings and continuing to fix them?

Jacques Gilloteaux, is this the paper you were referring to "Cryofixation of basement membranes followed by freeze substitution or freeze drying demonstrates that they are composed of a tridimensional network of irregular cords." Chan FL, Inoue S, Leblond CP 1993?

In regard to using microwave acceleration, I am a bit puzzled because I've performed a few trials processing tissue for EM ultrastructure in parallel with and without microwave aid and the former samples are usually slightly better preserved than the latter. Some of the images I posted come from samples processed with, some without microwave, and the problem seems not significantly different. I'll keep trying both ways.

Hi Nicola, according my opinion, the problem might result from the time of fixation. I fix small rat aorta rings for 3 hours at 4 degree of Celsius. Ludmila. 

That sounds like a shorter time in comparison than 2 h at room T, correct?

I think you should remove the fixation of 2.5 % glutaraldehyde 12-24 hours. 

I'm using  1% OsO4 ....

I thought to use longer fixation than 1.5 h, we have good experience to fix aortic rings for 3 h. I also use only 1% OsO4.