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One of my first experiments in graduate school was watching Drosophila 
S2 cells bleb after fixation with PBS/4% PFA for 10 min, I highly 
recommend it! From what I gather, the reason blebbing happens is the 
decoupling between the fixed cortical actin network and the unfixed 
(because it is mostly lipid) cell membrane (see 
doi:10.1016/j.fob.2014.02.003). At least in these cells, I observed it 
happening almost immediately after the fixative is washed off of the 
cells. During the fixation there were no morphological changes, but as 
soon as I washed it off and replaced it with PBS, the cells began to 
swell evenly, and after 15-20 min, large blebs formed with a 
simultaneous relief of the swelling.

I have observed the same effect in pretty much every cell line I've 
used, including adherent and suspended cells. I have found that 
introducing a 10 min permeabilization step immediately after fixation, 
e.g. with 0.2% Triton X-100 or (even gentler) 40 ug/ml digitonin, serves 
to prevent bleb formation, and makes for much nicer-looking cells. I 
don't know whether there would still be loss of volume, though.

Most people I went to for help with this issue thought I was crazy, but 
it turned out that they were all permeabilizing their cells for staining 
purposes anyway, while I was not permeabilizing because I was using 
cytoplasmic dyes and fluorescent protein tags exclusively, hence they 
never noticed any blebbing.

Mel Symeonides

On 12/9/2014 8:49 PM, James Pawley wrote:
>
>>
>> Cells on coverslips tend to flatten during fixation.
>> In LM the Z resolution is always poorer than in XY and shrinkage in Z
>> exacerbates this mismatch.
>> Does anyone have any suggestions for preventing or reducing shrinkage.
>
> Dear Jeremy,
>
> I have never liked to use the word "fixation" in this way. It implies
> that it stops changes when in fact it doesn't. It just stops the
> specimen from rotting right away. How about chemical treatment?
>
> It should perhaps not surprise us that, on encountering the fixative,
> living cells don't say to themselves "Oh, hurray, I'm going to be in a
> famous photo on the cover of Nature!". Instead they react in a variety
> of ways (blebbing, retraction etc.) in an attempt (one assumes) to
> avoid being killed. When the cells in question are only one cell-layer
> quick, diffusion is fast, the struggle doesn't last long and the
> rearrangements, if not always minor, are at least usually fairly
> obvious (blebs on white blood cells: they look like bubbles on the
> outside). In fact, you can (and should?) watch this process on the
> stage of a phase or DIC microscope.
>
> However, it is not uncommon for "fixation" to be followed by some sort
> of dehydration (into a graded series of ethanol or acetone and then
> perhaps into wax or a clear embedding liquid) and as the structural
> shape of both lipids and proteins is largely determined by their
> interaction with the surrounding water, it is this process that
> produces the greatest distortions (a minimum of 60% volume shrinkage
> in soft tissues such as embryos, according to the most careful studies
> by Alan Boyde at UCL, London.). When applied to cells attached to
> glass, the glass acts as sort of a "drying frame" (i.e., like those
> used to prevent the pelts of fur-bearing animals from shrinking as
> they dry), and by preventing the shrinkage in the x-y direction,
> increases that in the z direction.
>
> Fixation methods that start with a freezing step show some improvement
> as long as they occur fast enough to preclude the formation of ice
> crystals. Impacting the cells on a Cu block cooled with liquid He
> avoids ice crystals for the outer 10-15 µm; using a high-pressure
> freezer that pressurizes the specimen to about 2,000 atmospheres
> before applying the LN2, can extend this to over 100 µm. However, even
> going to these tedious (and expensive) lengths will prevent only some
> of the shrinkage attendant on replacing the ice with some non-polar
> solvent (freeze-substitution of the water by acetone containing OsO4).
>
> Which is why many folk replace the water with glycerol, which is as
> hydrophilic as water, although a bit lumpier.
>
> Chapter 18 in the handbook of Biological Confocal Microscopy (3rd
> edition) goes into some detail on the subject.
>
> Regards,
>
> Jim Pawley


-- 
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
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Phone: 802-656-1161