3 replies to this topic

[aztecan princess]

I’m new doing immunofluorescence:

Do you made fresh PFA 4 %, PBS-Triton and PBS-tween?? Or could be stored?

I read PFA could be stored at -20°C, and defroze just ones. But what about PBS-tween and PBS-BSA-tween?

For a cell culture immunofluorescence, protocol says: fix with PFA 4 %, but a labmate told me to use PFA 4%, then EtOH, then acetone.
Which is better and why?

Thank you very much for your advice!!!

[lotus]

I have always used freshly made 4% PFA. There are 40% PFA bottles commercially available that can be stored at Room temp. You can dilute it 1 :10 just before use. This will take only a minute and can be done each time. PBS- tween, triton etc can be stored, don't worry. Just make sure there is no contamination. Bacteria love to grow in PBS. If you add BSA to PBS, then store at 4 C. BSA will degrade over time, so use it up quickly. There are many procedures for immunofluorescence and it depends on what your goal is. After fixing, you can wash off in methanol and permeabilize with triton if you want. You need to read up different protocols and find out what each reagent is used for before designing your own.

[bob1]

Anything with a detergent in it on the PBS line of things is very likely to be OK in the fridge. BSA-PBS can be frozen with no problems, as can 4% PFA, though the PFA should be used in less than 6 months.

[toprak]

we always make a big batch of 4% PFA and then aliquot it (10ml & 50ml) and we store them at -20 for months. I don't refreeze them more than twice.