Hi All,
I wanted to know if suspension cells can be stored at 4 degrees after fixation with 4% PFA for 20 minutes. After good washing if I store them for around a week at 4 in PBS, would it have any impact on cytospin, IF or IHC.
OR
should they be processed in eppendorf for complete staining and then stored at 4 before cytospin.
Any help would be appreciated.
Hi Sheerien,
I've never done this with suspension cells but we fix liver and skin tissue pieces in 4% PFA over night and then, if not immediately processed, either keep them in PBS at 4°C for a few days or start the dehydration step for embedding and then keep them in 100% EtOH until further processing. Both IF and IHC work perfectly well.
Hope this helped for a start.
Sheerien,
PF cross-links proteins but does not stabilize lipid in membrane. As a result, cells fixed in suspension will maintain fairly good morphology when stored for an hour or so after fixation and in the cold. They will be less stable if stored in 4% PF for hours or days and quite unstable if fixed and then stored in buffer for days. During storage, cell morphology will deteriorate as lipids in membrane bilayers destabilize and dissociate.
As Eric indicated, pieces of tissue and organs are much more stable than isolated cells after fixation in PF, although their structure will also break down over time especially if they are stored in buffer.
Hi Robert,
Just for my understanding, normally tissues in Histo path are fixed in PFA, some times for 2-3 days. when they are immuno stained, the morphology of membrane and cells seem to be quite good. Why doesn't lipid destabilizes in this case?
Sheerien,
For standard tissue histopathology, there is much more protein present than with isolated cells. This includes serum proteins and ECM proteins like collagen and elastin which tend to stabilize gross tissue structure when they are denatured and crosslinked by PFA. But, more importantly, tissues and organs are processed through an alcohol dehydration series and (usually) xylene before embedding in paraffin. Alcohol, especially at high concentrations, is an excellent protein denaturant which further stabilizes tissue structure and then xylene extracts all lipids from the tissue. So In standard paraffin tissue sections, all lipids have usually been extracted, but this occurred after tissue structure was stabilized by fixatives and alcohol denaturants.
If you look at tissue fixed with PFA alone using electron microscopy, membrane structure and general cell architecture are actually highly disrupted. This is the reason why much better cross-linkers like glutaraldehyde are used for electron microscopy and agents that stabilize some lipids like osmium tetroxide are used to fix samples for EM.
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