Sucrose acts as a cryoprotectant by removing the water (by exosmosis?) Does this alter the size of the cells? Technically the cells should shrink due to the 30% sucrose hypertonic solution being used.
I realize that sucrose is removing the water hence, prevents the formation of ice crystals and thereby maintaining the cellular architecture but doesn't exosmosis damage the cellular architecture at the same time?
Also, would an observation such as cellular swelling/shrinkage be considered significant/reliable when sucrose itself can have such effects?
Does the 15-20-30 gradation prevent such effects? If yes, how?
There would be a huge change in cell volume in live cells; but there is no significant damage to the membranes and water will get in and out without any major disruption to the fixed tissue. Even if there is small change in cell volume during sucrose treatment it will recover when you put your tissue in PBS after sectioning. Therefore when you image your cell sizes treated with sucrose (freezing) or without sucrose (unfrozen vibratome), you will not see a big change in the cell soma size or volume.
best wishes, Refik
Dear Vasishta,
I use sucrose both for cryoprotection of fixed tissues and also for protection of live brain sections where I replace extracellular Na+ with sucrose (for details see article below). In your case in fixed tissues there is no significant damage to the tissue or the cellular walls as water moves via channels on the membranes which are stabilized by fixation. After cutting your sections you incubate your sections in PB saline again and the water moves back into the cells with stabilised membranes and therefore due to membrane stabilisation shrinkage or swelling of tissue or cells are minimised. It is almost filling a solid container (not a balloon) with water or emptying it. However, there may be more shrinkage due to the fixation process itself. I can't exactly recall but some older studies back from 1980s reported such shrinkage of cellular soma of ~5-10% due to fixation. For use of sucrose in live brain slices see my publication below.
Article Neurobiotin Electroporation for Combined Structural and Func...
thanks! But does it mean that water is exiting the cell without leading to a change in the volume?? Or is it that the fixative has stabilised (definition?) the membrane, which allows water to flow in and out without damage?
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