Dear all, my question is as above for immunofluoroscence staining purposes. Thank you for your kind responses.
Justin, for an analogy, please cf:
https://www.researchgate.net/post/4_paraformaldehyde_for_fixing_cells-4_degrees_or_room_temp.
"it depends"....
After fixation of your cells with 4% (hopefully 'buffered') PFA-solution NOT ALL organic / inorganic substrata of your cells perhaps are "really fixed"(in terms of irreversible change of conformation of proteins, like using glutar-di-aldehyde for primary fixation, etc.). So you should inform about the task of your research experiment /processing - your IF-staining - a bit more in detail.
Usually (and IMHO) for IF-methods there should not be a very big difference in using PBS at room temperature...assuming you use the correct PBS (make, osmolarity) (:-)) but perhaps a literature search in depth for the matter would find some argument to do washing either with ice cold PBS or PBS at normal room temperature.
PS: the purpose of "washing fixed cells" first of all is (as the term "washing" = get rid of, cleaning normally implies) to get rid of (most probably otherwise disturbing) remnants of unreacted fixative and or byproducts of the fixation reaction itself.
Coming also to mind: additional 'blocking of free aldehydes' in a tissue section or fixed cell suspension, since such left aldehyde sites would interfere with (dye, staining) reactions after further processing....
Hello,
I have performed ICC/IF on several cell types, including primary cells and cell lines. And I usually got good images by using PBS at RT for washing.
Good luck!
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