We have recently experienced some problems with frozen tissue samples being damaged at some point before they are sectioned. We are looking at teeth that have been fixed in aldehydes then decalcified in formic acid/formate before freezing and sectioning. I suspect that the culprit is either the freezing procedure or the storage of the frozen blocks. The tissue that has disappointed us was frozen in OCT slowly in the cryostat and then stored there. I realize that for unfixed tissue 'snap' freezing is usually advocated but thought that gradual freezing was kinder if the tissue had been stabilized by chemical fixation. I realize this is a nieve point but we may be missing something obvious. I would appreciate hearing what other people use even if it is mundane and routine.
Rex Holland
rholland@umich.edu

Rex,
What is the damage and which type of microscopy are you doing? The problem might be the formic acid solution, very very rough on tissues. Is the calcium all removed?
Ed Calomeni
emlab@opus.mco.edu

Rex,
Snap freezing will give better results even for fixed tissue. Fixation usually will further improve morphologic detail over that obtained with unfixed samples because of greater resistance to crystalization of fwater. Slow freezing allows formation of more and larger ice crystals within the tissue. For example, slow freezing of vibratome sections helps antibody penetration by ripping big holes in the membranes.
Although formic acid solutions are better than other acids for preservation of detail, you might experiment with warm EDTA for better results. Further discussion of decalicification techniques may be found in "Preparation of Decalcified Sections" by Edward B. Brain, pub. by Charles C. Thomas.
Glen MacDonald
glenmac@u.washington.edu

Although I am not an expert in the theory and methods of freezing biological tissues, I do know that the techniques used to prevent ice crystal formation are not trivial.
Vitrification (freezing without ice crystal formation), although theoretically simple, is not easy to achieve in practice if cryoprotectants are not used. The theory is to remove the heat so fast that the water has no time to form a crystal. To remove the heat this fast requires special equipment and very small specimens. Snap freezing, which I assume means plunging a specimen into a beaker of liquid nitrogen or pentane, is not usually sufficient to freeze large histological specimens without ice crystal damage. Neither will fixing the specimen prevent ice crystal damage.
The simplest solution for preventing freezing damage in routine histological specimens, which are to be sectioned in a cryostat, is probably to cryoprotect the fixed material before plunging in liquid nitrogen. Tokuyasu has shown that sucrose solutions that are over 1.6 M can be frozen by immersion in liquid nitrogen without ice crystal damage.
As an aside, I thought that the discussion about being able to quantify the number and size of ice crystals in frozen tissue was effectively closed in the 1980's by the work of Dubochet, Unwin and others.
My advice to any histologist worried about the enormous tissue damage that occurs when freezing fixed or unfixed specimens in cryogenic liquid, is to cryoprotect before freezing, then freeze by immersion in liquid nitrogen. Sucrose will easily penetrate fixed tissues but should not be used as a cryoprotectant for unfixed material.
Paul.Webster@quickmail.yale.edu

30% sucrose in PBS is a good cryoprotectant for lungs and would probably work for hard tissues, also. Fix the tissues first, then remove fixative in three changes of PBS (10 minutes each for lungs, probably longer for your tissue). Soak tissues in cryoprotectant at least overnight, at 4C. Blot off excess liquid, then snap-freeze the tissues in embedding medium. I found that tissues embedded in Lipshaw's medium were easier to section than those embedded in OCT. You might give the Lipshaw medium a try. I also concur with the comment about your decal solution - I think EDTA is considered to be a "kinder and gentler" decalcifying agent than acid treatments.
Good luck!
Jane A. Fagerland, Ph.D.
FAGERLAND.JANE@igate.pprd.abbott.com

I don't know if your interest in freeze substitution is to actually try it out before buying a machine but if it is, here is a neat way to do it. Fix the biological material (either by freezing or by chemical means).
If you choose chemical fixation you must then cryoprotect the material by infiltrating with 2.0M sucrose and then freezing on metal pins, by dropping them in liquid nitrogen.
Once frozen, transfer the material to dry methanol (just open a new bottle) which is being held in a tube, in a styrofoam box filled with dry ice. Keep the material in the cold methanol overnight. Remove the methanol and replace with fresh methanol, leave for a few hours, and replace with a methanol-Lowicryl mix (1:1). If you want to keep the tubes on dry ice you can use Lowicryl HM 23, otherwise, transfer the tubes to a freezer. Keep increasing the amount of resin in the tubes until you are in 100% resin. Leave overnight, replace with fresh resin and polymerize with UV light. Great embedding (I have pictures) with low cost.
Variations include adding 1% osmium tetroxide or 1% uranyl acetate to the methanol. The contrast is subtle but significant and the osmium does not affect polymerization or immunolabeling.
Paul Webster
Paul.Webster@quickmail.yale.edu

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