Hello, I need to stain some primary culture cells in permeabilized and non-permeabilized conditions to test if a protein is localizing to the exterior of the cell membrane. Our existing fixation protocol for this is to use ice cold 4%PFA for 5 minutes, but some lab members have had issues with the PFA permeabilizing the membrane. Does anybody have any recommendations for an alternative fixation protocol that would maintain membrane integrity?
- Use a lower concentration of PFA, like 2% or 1%, and fix for a shorter time like 2-3 minutes. The higher concentration and longer fixation can permeabilize membranes.
- Try using a different crosslinking fixative like glutaraldehyde at a concentration of 0.1-0.5%. Glutaraldehyde preserves membranes better than PFA.
- Methanol fixation at -20C can also work well. Fix the cells in 100% ice cold methanol for 5-10 minutes.
- Some antibodies may still be able to bind to their epitopes on non-permeabilized cells, so you could try skipping fixation altogether. Just block with serum and stain live cells.
- You can permeabilize after fixation with a detergent like Triton X-100.
The key is using very gentle fixation conditions to preserve the membrane integrity for the non-permeabilized cells. Lower PFA concentration, glutaraldehyde, or methanol at -20C are good options to try.
Hi,
I don't have experience with cell culture itself, but there are 2 methods you could try, which I know work on tissue sections:
You could try PLP fixative (Paraformaldehyde, Lysin, Periodate). The idea is that periodate oxidates certain sugars so they can react with the amino group in the lysine and be cross-linked with the paraformaldehyde. This has been shown to preserve membranes better (see e.g.:Article Periodate-lysine-paraformaldehyde fixative. A new fixation f...
;Article PLP fixation for combined routine histology and immunocytoch...
;Article Comparison of cell fixation methods of induced sputum specim...
).Alternatively you could try a stronger cross-linker. Glutaraldehyde has been suggested already, you could also try glyoxal (see: Article Glyoxal as an alternative fixative to formaldehyde in immuno...
).Hope this helps!
Cheers,
Sven
Hi Salah
For fixing cells under conditions that preserve membrane integrity, you can consider using 4% paraformaldehyde (PFA) chilled on ice for a shorter period of time, or even trying other fixatives like glutaraldehyde. Here's a basic protocol you can follow:
Alternative Fixation Protocol:
Using 0.1% or 0.25% glutaraldehyde in PBS is also an option that may be less prone to membrane permeabilization, although you may need to optimize the conditions based on the cell type and the goal of your experiment.
Always remember to work under cold conditions (4°C) to prevent membrane degradation and permeabilization during the fixation process. Additionally, consult with your colleagues or supervisor to see if there are specific protocols tailored to your laboratory's needs or the type of cells you are using.
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