Beat, Ummmm at the end of your message, you mention variable results with
paraformaldehyde on GFP. Could you elaborate? Also, If you dont use
Paraformaldehyde, could you tell me what you do use? if its
glutaraldehyde, why would this be better? and what concentration and time
do you use it? Im working with semi thick cryosections, b/w 10-20 ums.
On 28 Oct 1997, Beat Ludin wrote:
> Hi Neva
>> > The protocol for the 4% Paraformaldehyde (PFA) is as follows:
> >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.
>> Reduce the fixation to about 30' at r.t. for cell monolayers.
> However, pFA fixation has it's problems. At least in my clumsy hands it
> seems to be the one that produces the most artifacts on the subcellular
> level (it's ok for tissue preservation). A lot of cytoplasmic proteins
> can be partially exctracted during or after pFA fixation, especially from
> the thinner parts of the cell, and some proteins can even be de-localized
> (e.g. MAP2 falls off microtubules during fixation), so the fixed pattern
> does not always reflect the in vivo pattern. Furthermore, some cells that
> are plated directly onto glass (as opposed e.g. to poly-lysine coating)
> tend to come off. Last but not least, I found the preservation of GFP
> fluorescence rather variable. Personally I would recommend using either
> glutaraldehyde, EGS, or -80degC MeOH for fixation and conventional
> immunostaining with anti-GFP-Abs.
>> I guess I've sufficiently confused you now.
> Dr. Beat Ludin, FMI, Maulbeerstr 66, 4058 Basel, Switzerland
> Tel. +41 61 697 6697 / FAX +41 61 697 3976
> Internet:ludin at fmi.ch / Compuserve:100102,1527