Neva Morales wrote:
> Hello everyone,
>> I need some help. Can anyone point me to a complete protocol that
> thoroughly explains how to make a paraformaldehyde solution. I'm trying to
> fix GFP cells with paraformaldehyde and I ran into some problems; I lost
> most of my cells and there was a lot of junk on my coverslip.
The protocol for the 4% Paraformaldehyde (PFA) is as follows:
A. Phosphate Buffered Saline (PBS):
Melt: 0.76% NaCl, 0.1% Na2HPO4, 0.047% Nah2PO4´2(H2O) in sterile water (DDW).
Adjust the pH to 7.2 using NaOH.
B. 4% PFA fixative.
Melt 4% PFA in PBS at 60 degrees Celsius for 1-2 hours. Cool it down to room
temperature and readjust the pH to 7.2 with NaOH. You should get a clear
solution. Freeze and store the fixative until use. Freeze fixative (at -20
degrees Celsius) can be stored for at least one year.
After unfreezing the fixative, fix the tissue for 1-3 hours at room
temperature (depending on how thick is your tissue), or for over night at 4
degrees Celsius. When fixation is done, wash the tissue twice, 10-30 min.
each, with PBS.
I hope that will help.