A standard protocol for intracellular staining usually separate the fixation and permeabilization part.
Can I do the fixation and permeabilization at one time by incubating cells with 3.7% formaldehyde with 0.1% Triton-X in ice for 30mins?
I include 0.2% TX-100 in my blocking buffer/immunofluorescence diluent. It seems to work well, no permeabilization necessary (as you permeabilize during the blocking/staining).
My immunofluorescence/blocking buffer recipe is PBS with 2% normal goat serum, 0.2% triton x-100 and 0.05% sodium azide.
Combining fixation and permeabilization should work, as it's done with much gentler detergents and formaldehyde for FACS (see BD Cytofix/Cytoperm).
Hi Ki Siang Lim
Mixing of formaldehyde with permeabilizing agent is in fact unnecessary. Formaldehyde is quite a small molecule and is able to crosslink proteins on its own (fixation step).
It is for the antibody binding step, permeabilization is required. In my experience diluting the antibody in a mixture of 50% blocking solution and 50% 0.1% Triton-x-100 (in PBS) provides good enough blocking and permeabilization effect. It gives very good staining results.
Good Luck!
U may fix the cells on cover slip using 4% para formaldehyde , for 15 mins @ room temp, followed by washing and permeablization using 0.05-0.1% tritonx 100 or what u can do -if you want to stain a nuclear antigen u can directly use acetone:methanol(1:1) mixture for fixing as well as permeablize(@ 0 degree for 10 mins), no seperate step for permeablization is required or you can use methanol for both the purposes.
all d best.
For the regular action of fomaldeyhe the pH of the solution has great impact (7,0-7,6). It would be interesting how the Triton changes pH. At a pH above 8 it doesn't really work.
This is my protocol:
3.7% Formaldehyde + 0.1% Triton-X in 1X PBS..
Incubation period = 20mins at 4 Celcius
I also use the BD cytofix and cytoperm and follows its protocol..
I have lost a significant number of cells when i use my own protocol but I can get sufficient cell numbers if I used BD cytofix and cytoperm..
the centrifugation force = 2200rpm for 5mins at 4 Celcius..
I have no idea why 3.7% formaldehyde + 0.1% triton-X can cause significant cell loss..
should I increase the centrifuge force? Please help. thank you
Hi,
It may have to do with the formulation of cytofix/perm as compared to your solution
Cytoperm uses saponin as the permabilizing agent which tends to be thought of as a gentler detergent than TX100 is. It may be that your cells are sensitive to triton x 100.
Another possibility is to fix in methanol which will permeabilize and fix your cells but you may encounter the same cell loss issues. Methanol also extracts a lot of cytosolic proteins (PFA + triton at the same time may result in cytosolic protein extraction).
Finally, are you sure that your formaldehyde is pure? I haven't had many issues with using regular formaldehyde stabilized with methanol but in some specific instances have found that using PFA or other, purer reagents has improved my protocol.
It is advisable to keep the fixation and permeabilization steps separate.
Fixation causes crosslinking while pemeabilization dissolve lipids making the cells permeable to flurophores or antibodies. So mixing both the steps can hamper the final outcome, if you want to save time rather it would be better to reduce duration of each step rather than clubbing the steps.
For fixation you can use 3.7%-4% PFA in PBS/MilliQ, and for pemeabilization 0.1%Triton X is appropriate.
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