Kashish,

You already may be aware of this review which mentions fluorocarbon-based oxygen carrying fixatives:  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1915585/.

Hope it helps,

Michael Delannoy

Thanks Michael!

Yes, I have read this review but I couldn't pick out specifically which TEM approach I should follow as the review clearly stated that TEM cannot be used in in vivo situation. If you suggest fluorocarbon-based oxygen carrying fixatives, then I will definitely read into it.

I've worked with these people (but there are others):

Klaus Qvortrup (Copenhagen)uses perfusion fixing with add oxygen and the tannic acid stain (the fluorocarbon you were mentioning). It is very very hard and the glycocalyx structure comes out short and blobby, but with excellent coverage.

Chris Neal uses a variety of methods all with perfusion fixing: Alcian blue is ok but is not that dense but can be seen in light so helpful in location of the part of interest. Also the Lathanide techniques BUT these can precipitate so be careful else you just look at crystals.

With all processes careful control of pH and ionic concentration is vital so it is best to talk with them direct.

Hi Kashish,

what kind of material do you want to visualize? In our group (Chappell, Jacob, Becker) we are using a lanthan based fiaxtion technique...

Please feel free to contact me!

Best wishes from Munich,

Florian

Kashish,

How about staining for proteoglycans? Isn't the glycocalyx a membrane bound proteoglycan?  This paper:

http://onlinelibrary.wiley.com/doi/10.1002/ar.1091870404/pdf

uses either safranin O (with fixative in phosphate buffer) or ruthenium red (with fixative in cacodylate buffer). 

hope this helps,

Michael Delannoy

Hello again Michael,

Yes, I have read some research that used ruthenium red to stain glycocalyx but the thickness of glycocalyx obtained from this method seems to be quite underestimated as compared to other modified TEM methods like with Alcian blue or with rapid freezing/freeze substitution.

Ruth Red was used extensively prior to the early 90's for glyc staining. I'm not really sure why it is not popular any more. I don't know much about safarin O but it does not have a heavy metal in it so to get density it would need other stains to bind to it, perhaps this is why it has not been used much for EM glyc imaging.

Just thinking out loud here,

I know that tannic acid acts as a mordant and binds more uranyl acetate in subsequent staining steps, so you get more intense staining of cytoskeletal filaments..  Perhaps tannic acid in the fixative followed by safranin O?

I have used various lectins in lectin-cytochemistry to demonstrate the modifications of carbohydrates in the mouse zona pellucida during folliculogenesis and after ovulation. Although your research interest focuses on the blood vessels, the procedure is very similar and should yield good lectin-labeling results in TEM.  You might want to take a look at the following three references: Histochemistry and Cell Biology (2000) 113:207-219, Histochemistry and Cell Biology (1999) 111:23-32, and Molecular Reproduction and Development (2001) 60:517-534.

The main problem is it being in the vessels when you get round to it as it sheds. Klaus Q gets amazing glycocalyx or 'surface coat' by perfusion fixing without any gap, ie fixative straight in without a flush. Tannic acid is a mordent and I think it is good at binding the GLX to itself so it does not wash off in the embedding (no need for the Saf O as the UA binds happily it it)

I'm getting (with the Bristol Renal group) good results for quantum dot stained lectins, the advantage is it seems to work on horrid parafin fixed sections converted to EM. Of course you can;t see the structure.