Hi all!
Can a mouse whole vertebral column be stored in 4% PFA for longer than 24 hours, but shorter than one week?
I plan to put the vertebral columns of my mice in PFA on a Friday and do sucrose the following reagents on Monday. Will this affect the quality of the extracted spinal cord later on?
Thanks in advance for your help.
Cheers!
Hi Alaa,
In my opinion you can store the tissue in 4%PFA for 48 or 72h.
Hi Alaa,
In my opinion you can store the tissue in 4%PFA for 48 or 72h.
You want to avoid over-fixation if you want to do IHC for a scarce target, but weekend-fixation is fine for most IHC work and sustaining a lab/life balance.
May by you should do a few test runs before you spend all the samples and find out that IHC or other staining are not working.
In general it should be OK. Although my suggestion is to use the good old '10% buffered formalin'. Formalin would avoid the 'hardening' effects of Paraformaldehyde for weeks, one reason is that it contains 10-15% methanol. If the formalin is good (no residue at the bottom of the bottle) then I think it is as good as PFA, if not better.
Regards
It may be OK or it may be not: it depends on what is your purpose later on. For example, if you need to do immunostaining, a long fixation is required by some antigen/antibody complexes whereas it is detrimental for others.
Best regards
Stefano
Yes, the above replies are correct, in that you should be fine. If left too long (several weeks) then some deterioration of tissue can occur since PFA can eventually change to formic acid and cause some holes in the tissue. Refrigeration can delay this process so you can put fixed samples in the 'fridge over the weekend. Should your tissue be fixed too long and you are conducting an IHC process, an antigen retrieving technique can then be used.
It is way better to perfuse mice/rats with heparanized saline first, then with 4% PFA, extract the spinal cord and wash it for a couple of days in 20-30% sucrose. Perfusion with PFA allows an homogenous PFA fixation in the spinal cord, which is not the case with post-fixation, even worst if the spinal cord is kept inside the column...
Sighild
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