Dear Colleagues
Hi,
I grow Hep2 cell line and am going to fix it. But I am wondering which method is better for ANAs (antinuclear antibodies)? Fixation with paraformaldehyde or glutaraldehyde?
Would you please tell me something about your experiences?
with the best
Amir
I have not heard much about using glutaraldehyde. Check the website below for the popular cell fixation protocols. (IHC world is a great resource). It is always best to try a few if you have doubts about antibodies. However, your antibody spec sheet should tell you how the cells were fixed for IHC. If not, you can call the manufacturer and ask for their fixing protocol so you can reproduce it.
http://www.ihcworld.com/_protocols/general_ICC/fixation.htm
There isn't much detail of the techniques and approaches you plan to use, so it's hard to give you a definitive answer. However off the top of my head I see no valid reason to use glutaraldehyde for fixation of cells on which you will be doing some sort of immune-based detection technique. Glutaraldehyde is usually reserved for electron microscopy preparations, because it preserves cell ultrastructure better than formaldehyde. However the protein cross-linkages induced by glutaraldehyde are more extensive and considered largely irreversible, compared to those induced by formaldehyde, which can be reversed with heat pre-treatment. This makes glut an unfavorable fixative for immunofluorescence or immunohistochemical techniques.
Use paraformaldehyde. Glutaraldehyde is important for fixation at some other techniques. But for your antibodies, it is very unlikely to have any result with glutaraldehyde.
Use 4% PFA at 4 degrees for 10 minutes.
If it doesnt work, then use methanol fixation at -20 degrees for 10 minutes as another method of fixation.
I have read some recommendations for using glutaraldehyde for fixation of small molecules such as GABA, glutamate, dopamine etc. because these transmitters can be easily washed out from cells during permeabilisation stage. To prevent excessive formation of cross-links, it is better to use an appropriate blocking solution after fixation stage. Some researchers advice to use ethanolamine or Tris for these purposes.
We used in our laboratory mixed solution of 4% paraformaldehyde +0.25% glutaraldehyde for immunocytochemical staining of hippocampal cell cultures with antibodies against GABA. Indeed, the quality of staining dramatically increased when we used the above-mentioned fixative solution with glutaraldehyde. It was possible to detect GABA distribution along dendrites and axons of GABAergic neurons. In the case of 4% PFA without glutaraldehyde, we can see weaker fluorescence of antibodies only in the soma of neurons. When we stained our cell cultures with antibodies against calcium-binding proteins or glutamatedecarboxylase 65/67, we did not see any differences after addition of glutaraldehyde to the fixative solution. Thus, it is better to test both PFA and glutaraldehyde to choose an appropriate technique.
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