[Histonet] PFA degradation

[Emily Sours]

>
> My question is...how do others commonly deal with PFA??  Do you make it
> fresh each time you use it??  If so, what is the favorite protocol??
> And, just out of curiosity, has anyone ever done an analysis of stored
> PFA to see how fast it breaks down...or how long it can safely be stored
> and used??

We used to make 4% para in PBS, then freeze aliquots until we needed
it.  It always did the job well for embryonic chick tissue.  Now we
only use 4% para 24 hours after it was made according to Tom Jessell's
protocol.  (We switched to his because we received antibodies from him
and wanted to make sure we followed his protocol completely.)
I would love to know if someone has analyzed the degradation of para.
My guess has always been that if it's not precipitated nor has
something growing it (that would signal contamination that boggles my
mind), it's good.
I've also found that people in the lab like to blame para for the poor
quality of their sections (quality of tissue and of staining).  That
isn't the problem; someone just didn't fix long enough or can't
section well, etc.
our old protocol:
Boil 400 mL water with stir bar.
Add 20 g paraformaldehyde, stir for around 5 min.
Clear with 1N NaOH (I always use 12 drops which makes the pH perfect)
pH to 7.2-7.5.
Store at 4C or freeze into aliquots.
Jessell protocol:
http://sklad.cumc.columbia.edu/jessell/resources/protocols.php


Emily
-- 
fortune smiles on the brave and spits on the coward
--aguirre, wrath of god