Freeze Substitution to Retain Soluble Components

Greetings all!
I am trying to use freeze substitution to retain soluble oligosaccharides in plant tissues, and am not yet convinced that I have been successful, partly because of embedding/infiltration problems (I think!). We have tried a variation of the tannic acid fixation with Kaeser's substitution cocktail, and stuff seems to look ok (tough to really assess when the scope has been down, unfortunately), although we have been advised that probably LR White is easier to work with than Lowycryl K4M, which is what we used the last time, and it doesn't section happily. Would like to try another run (just got some fresh LR White) and thought it would be useful to ask for some input from the group. It's probably of interest to more than me, but if folks prefer to respond directly, I will summarize the responses and post them. I'm certainly looking forward to your input!

S.Shea Miller
MILLERS@em.agr.ca

Dear Shea,

Have you considered fixing the tissue in aldehyde and embedding in one of the other (hydrophobic) Lowicryl resins (e.g. HM 20, HM 23). They polymerize at lower temperatures and cut very well. We have had some success with soluble proteins in the pancreas and there are published examples of this method.

Here are a few references which might be of interest: van Genderen et al, 1991 J. Cell Biol. 115:1009-1019. Oprins et al, 1994 J. Histochem. Cytochem. 42:497-503.

There is also a recent paper in Microscopy Research & Technique which covers this subject but omits to refer to the above papers. The paper is in MRT, 1996 33:251-261. Look too at the work by Humbel & Schwarz which is cited in the MRT paper.

Paul Webster
Paul.Webster@quickmail.yale.edu

Dear Shea,

Low temrerature embedding/infiltration is not an easy procedure. The concetration and the distribution of the soluble proteins play an important role of successful LT embedding. I have had some success to retain soluble proteins in different type of plant tissues. Enzyme activity in vacuoles and in cell wall,using post-embedding techniques is our interest. Lowicryl resins: HM23 and K4M, as well as LR White were used at different temperatures and times (-80 to -20C; 2days to 2 weeks) after "mild" GA fixation OR cryo- fixed plant tissues, using PLT techniques (dehydration at progressively lower temperatures). LR White, after UV polymerization developed very small air (?) "packets". It doesn't section happily, yet not a major problem. "An Osmium-free Method of Epon Embedment That Preserves both Ultrastructure and Antigenicity for Post-embedding Immunocytochemistry", Phen, K.D., Rustioni.A., and Weinberg, R.J., J. Histochem.Cytochem. 42, 1995. You may adopt this procedure to LR White. It should work well. Good luck! Any other ideas, please let me know.

Laszlo J. Veto
VETO@BCRSSU.AGR.CA