I want to know whether or not standard acetone fixation (10 minutes at 4 degrees celcius) sufficiently permeabilises the cell membrane to allow binding to focal adhesions for visualisation using immunofluorescence. The focal adhesions in question are Talin and Vinculin.
If anyone has any experience doing this, please let me know.
Thank you in advance.
In order to permeabilize cell membranes you have to use Triton X 0,2% or other detergents like Tween for 10 minutes after fixation.
Methanol -20 overnight fixation/wash with PBS/and permeabilize with PBS+BSA+TritonX-100 (0.3%) 30 mins.
Thanks to you both. I have tried the tween permeabilisation step and I use tween with TBS during each wash phase to ensure the membrane is very permeable but these two factors do not seem to increase the staining for focal adhesion proteins.
I will try the methanol fixation but I have heard conflicted opinions which state that methanol is actually poor for antigen preservation: http://medicine.emory.edu/documents/mimcore/immunostaining-protocols.pdf
Have either of you specifically stained for focal adhesions?
Thanks again.
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