Hello, I am trying to fix primary cells with glutaraldehyde. I am trying to stain cell membrane proteins and I have not been successful with PFA and methanol fixation. Does anyone have a protocol for glutaraldehyde fixation (conc., time, quenching etc.)?
Thank you!
Glutaraldehyde fixation is commonly employed for TEM and it is not very good for immunostaining, because glutaraldehyde makes more protein-protein crosslinks and therefore hides antigens from antibodies. If you do not see IF signal, maybe the issue is in antibodies you use, not fixator? Or your cells are destroyed after fixation? Specify your protocol and the problem you encountered more precisely, please.)
Glutaraldehyde is also horrendously fluorescent if you are using Immunofluorescence. Fixation with solvents will likely remove membrane proteins along with lipids. If the membrane proteins have an external epitope try staining live cells. Note that IF methods also typically include detergents to that may also remove membrane proteins.
Snezhanna Medvedeva My staining protocol is:
(Staining in primary astrocytes)
1. Fix with 2% PFA 15min
2.Wash PBS 3x5min
3.Block 10% NGS
3. Primary incubation overnight in PBS-BSA
4. Wash 3x10min PBS-BSA
5.Secondary incubation in PBS-BSA
6. Wash 2x10min PBS-BSA
7. DAPI staining
I have attached the imaging results below. As you can see the red signal is low and there are a lot of dots. Some people suggested using low concentrations of glutaraldehyde to fix.
Jiaqi Zou Oh, I see. I work with intestinal primary cells and stain membrane proteins as well, so I guess my advice can help. Firstly, usually people use 4% PFA (do not confuse with formalin, formalin must be 10%) and incubate for 30-60 minutes. Secondly, it's recommended to use permeabilizing agents (Triton X-100 or Tween 20) even for membrane proteins, since antigen your antibody binds could be located in cytoplasmic domain of the transmembrane protein. I add 0.3% Triton to my blocking solution (PBS+0,5% BSA+0,3% Triton X-100) and use it for antibody incubation too, both primary and secondary. Finally, the possibility remains that your primary antibodies are not working or pair primary-secondary is badly selected. If you wish you can check the specifity with WB. Or stain your cells with antibodies which were confirmed to be working, it will help to understand, where the problem is. To me, on this picture antibodies just don't bind antigens, but maybe adding Triton will help.
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