Hi Doug,
It probably depends on the level of resolution required which in turn determines the degree to which the available “fixable” structures in a cell must be tightly crosslinked.
I tend to be a bit on the OCD side when it comes to handling/preparing cells for microscopy. I suspect that super resolution techniques will find evidence of slight differences in fixation that might not be noticed by looking at things like the morphology of filipodia, etc.
I can’t comment on precisely what the effects of less than optimal fixation look like, but I always made up fixative fresh from desiccated paraformaldehyde and would only use that same day.
Formaldehyde solution begins to polymerize immediately. Formaldehyde forms so-called crosslinks of methylene bridges by reacting with amines, purines and several other moieties. These crosslinks become longer when formed by polymerized formaldehyde. My understanding is that this results in a less than optimal crosslinking arrangement, where adjacent crosslinked molecules may be spanned by longer crosslinks than might otherwise be formed. In other words, the various structures fixed in the cell are not as tightly “zipped-up” as they might otherwise be.
Much of the literature regarding formaldehyde is more relevant to and drawn from longstanding anatomical fixation techniques with very thick specimens (pathology), and doesn’t offer much help to high-resolution fixation of layers of cells or very thin tissue sections. You can get away with less careful chemistry when it comes to preserving organs and large specimens which fix for days vs. high resolution microscopy of single layers of cells or thin tissue which fix in minutes.
Jeff
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: Tuesday, September 20, 2016 10:34 AM
To: [hidden email]
Subject: aliquoting 4% formaldehyde
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I was talking with a student yesterday about their cells which did not look particularly good under the microscope. The discussion eventually came around to fixation. The student told me that he made up some 4% formaldehyde fixative and then froze back (20 deg C) aliquots for later use. This was something he had seem on an online research forum. In my fairly lengthy experience I have never heard of this being done and I am suspicious that this is NOT a good idea. Thoughts?
Doug
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