I'm brand new to EM and so I'm planning TEM experiments on embryonic day 18 to post-natal day 14 mouse lung. I am interested in glycocalyx of a specific cell type (Prox1-positive).

For glycocalyx, most protocols that I have found use a 0.5% Alcian Blue 8GX in with the 4%PFA/1%GA/PBS fixative. Next there is a 1% OsO4 + 1% Lanthanum (III) Nitrate immersion for the impregnantion step. Then, I will incubate with 1% Uranyl acetate before dehydrating and infiltrating with the resin. My question is whether this process is compatible with post-embedding immunogold labeling of the PROX1 which is a transcription factor in the nucleus, since I want to be certain that I am comparing glycocalyx on this specific cell type. Would staining adjacent thick sections be a better alternative. Perhaps you could advise me toward a better/more simple/straightforward method to show cell-type specific glycocalyx with EM?

Hi, Timothy, Alcian blue will expectedly label acid glycans, ruthenium red could be considered as well. I am afraid however that OsO4 may interfere with your ICC protocol - changes in protein conformation are possible. You could try post-embedding periodic acid/thoisemicarbaside-based polysaccharide labeling techniques AFTER you perform your ICC

I agree, OsO4 might interfere with labeling of your sample. But this will have to be determined experimentally. Immunogold labeling will be the most specific. You must remember to have adequate controls in place, to check for non specific binding of your antibodies to other antigens in your sample. If you have access to high pressure freezing and freeze substitution equipment, I would rather use these, bypass OsO4 and embed in LR White. Hope this helps.

You might want to take a look at this reference for tissue processing for immunogold labeling: Bendayan, M., A. Nanci, F.W.K.  Kan  1987.  Effects of tissue processing on colloidal gold cytochemistry.  J. Histochem. Cytochem. 35:983-996.