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pFA fixation (was:hello/help.)

Beat Ludin ludin at FMI.CH
Tue Oct 28 08:08:25 EST 1997


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.

Beat




-----------------------------------------------------
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




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