hello/help.

[cinnamon yuval]

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.
>
> thanks,
> nm

Hello.

  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.

C. Fixation.

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.
Yuval