[Histonet] Nestin and perfusion for spinal cord

From:"Donna Harclerode"

We have had no success with Nestin from Chemicon (Neu N either) and
would appreciate anyone having an antibody that works to share the info.
We are currently trying on chamber slides so they should, but do not
stain anything on the slides.  We used 4%PFA (from concentrate for 30
minutes to fix human cells.  Other antibodies work great.

For perfusion I always used freshly prepared filtered 4%PFA in PBS
unless there was a reason not to. I have also used the zinc formalin
solutions with good results.  I would not use NBF as it has methanol in
it to stabilize the solution and the pH is not always controled.  There
is a concentrated solution of PFA (up to 32%) that you can get from
Electron Microscopy Sciences that you can order through VWR that
probably would be ok.  I never let PFA solutions heat over 65oC
(breakdown of aldehydes) and it is a time consuming and not so nice
solution to make.  Be sure to use prill (Electron Microscopy Sciences-
very inexpensive and high quality) or granular if you are making the
solution from scratch as the powdered PFA gives a lot of dust and gives
you more exposure to the paraformaldehyde. Some antibodies preferred
2%PFA and a couple we used gluteraldehyde and PFA.  I perfused with RT
saline and then with cold PFA until the brain was white and the animal
(rat, pigeon or chicken) was very stiff using the left ventricle with
the right atria cut to allow the solution to leave the body.  We left
the brain and or spinal cord in the same fixative as the perfusion
overnight and transferred to 30% sucrose in PBS and left it in the
fridge for a couple more days.  This worked great for the majority of
antibodies on free floating sections. Lower concentrations of PFA gave
me soft tissue that was more difficult to handle for staining and

Thanks and good luck

Donna Harclerode, HT, (ASCP), HTL, QIHC
Cytori Therapeutics
3020 Callan Rd.
San Diego, CA 92121
858-458-0900 ext 322

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