RE: plugging holes in cells

Just to state that dissolving insoluble paraformaldehyde (white waxy
powder) gives you dissolved formaldehyde. Id is unstable under oxygen
forming formic acid. 
Fixation will normally lead to cell death / pore destruction that leads
to the uptake of small molecules like PI (680kD). You can probe with
bigger molecules to see if they stay out but sometimes molecules of
considerably bigger MW can wriggle their way into live cells.

Formalin is 35%formaldehyde stabilised in 15% Methanol

What do you want to achieve with the fixation? Can you not probe with
your small molecule prior to fixation?

Regards

Gerhard Nebe-von-Caron 
Research Scientist and Biomedical Engineer 
SPD-Spark
Swiss Precision Diagnostics 
Priory Business Park 
Bedford, MK44 3UP, UK 
Tel +44(0)1234-835474 
Fax +44(0)1234-835002 
mailto:g.nebe-von-caron@spdspark.com 
-----Original Message-----
From: Jayaraman, Sundararajan [mailto:anue2468@uic.edu] 
Sent: 04 January 2008 02:26
To: cyto-inbox
Subject: Re: plugging holes in cells


Perhaps fixation with 1% paraformaldehyde will help. For best results,
make a 5% stock solution of  paraformaldehyde by dissolving
paraformaldehyde powder in PBS. Heat the solution on a hot plate under a
fume hood since this produces harmful vapor. Bring to a boil. After a
short while, the powder will go into solution. Turn off the heat and let
it stir for about 10 to 15 minutes to completely dissolve. Filter
sterilize the solution and keep it in the refrigerator. Add 5%
paraformaldehyde solution to the cell mixture to a final concentration
of
1%. Fix the cells for 10 to 15 minutes at room temperature and you can
keep the fixed cells in cold for a day or two. This will fix the cells
without making holes unlike formaldehyde or glutaraldehyde.

Hope this works.

SJ

S. Jayaraman, Ph.D.
Associate Professor of Surgery
University of Illinois at Chicago
909 South Wolcott Avenue-704E MSB-M/C 790
Chicago, IL 60612
Phone: 312-355-5133
Fax: 312-355-1497

On Wed, January 2, 2008 12:59 pm, MODEL, MICHAEL wrote:
> Dear List:
>
>
>
> I am trying to do something to prevent penetration of a small (mw
under
> 1,000) hydrophilic molecule into chemically fixed cells. The cells can
> be fixed with formaldehyde or glutaraldehyde for example, but that
makes
> their membranes leaky, so I am looking for a way to either fix them
> without opening large holes or to somehow plug the holes afterwards.
> Does anyone have any experience that might suggest a possible
approach?
>
>
>
> Thanks in advance and happy New Year.
>
>
>
> Michael Model, Ph.D.
>
> Confocal Microscopy Core
>
> Dpt. Biological Sciences
>
> Kent State University
>
> Kent, OH 44242
>
> tel. 330-672-2874
>
>
>
>


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