Hi Sean,
Sorry for the delay.  

All the staining results I"ve referred to used 3% formaldehyde (before or after Ab treatment) which was made from powder no more than an hour before use.   I can't say what the mechanism of leaking is, but osmolarity and pH were specifically controlled for.  My guess is that the cross-linking, hence denaturation, of membrane proteins disrupts  the normal interaction between lipids and those proteins, and provides an access for larger molecules across the barrier.

I may not be clear on your procedure.  If you dissect the brains, you would expect an injury response, hence the problem with obtaining a good negative control.  When you refer to "live brains", is that tissue still in the animal? 

Overnight incubation of sample with Ab at 4C is a standard procedure in many cases and can give you good results.  The problem I see here is if you get good penetration of the Ab into the tissue, you will need to do substantial washing before fixing and adding secondary Ab.  That may preclude having live material in the end.

Please feel free to correspond off list.
Cheers,
C


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX 520-621-3709


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Sean Speese
Sent: Wednesday, January 05, 2011 10:50 AM
To: [log in to unmask]
Subject: Re: Question about surfactants to break surface tension

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Hi Carl,
  I just wanted to follow up with a few more questions about staining epitopes just at the surface.  My assumption was that the little bit of permeabilization that one can get from para may occur if you use 37% formaldehyde as your starting solution, as it has some low level of methanol.  However, if I understand you correctly, even high grade EM para will lead to some permeabilization? Is this due cells being osmotically shocked and lysed if the osmolarity is off, or some other mechanism?

 I would like to stain Drosophila brains to see if certain proteins are secreted into the ECM by glia and neurons after injury.  I could stain live material, but dissecting open the live brain is essentially going to elicit some type of injury response, thus making it hard to have a clean control.
Maybe I could keep the live brains that I am staining at 4 degrees to minimize any reaction from the dissection itself.

Thanks,
 Sean

On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) < [log in to unmask]> wrote:

> Hi Sean,
> Don't know of an alternative that can do what you want.  I would think 
> that the same properties of a surfactant that make it one would also 
> disturb lipid layers.
>
> Aside from that, I would caution against the assumption that fixation 
> without detergent allows only exterior staining.  The fixation process 
> will also open holes in membrane and allow Ab to penetrate.  At least 
> test it before committing to that route.  The best result I've had 
> restricting immunostaining to a surface is to stain live material, 
> then fix after washing.
> C
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> Univ. of Arizona
> 520-954-7053
> FAX 520-621-3709
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List 
> [mailto:[log in to unmask]]
> On Behalf Of Sean Speese
> Sent: Tuesday, January 04, 2011 5:44 PM
> To: [log in to unmask]
> Subject: Question about surfactants to break surface tension
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>   Bit of an off topic question, but we do lots of immunos on small 
> bits of Drosophila tissue that always float on top of the fix due to 
> the surface tension.  We can use low amounts of triton (.01%) to 
> overcome this, but I would like to be able to leave detergents out 
> until I am convinced the fixation is good.  In some cases I would like 
> to do staining of only things on the cell surface, and thus need to 
> leave detergents completely out of the staining procedure.  Therefore, 
> does anyone know of a good surfactant that would break the surface tension but not permeabilize cells?
>
> Thanks,
>   Sean Speese
>
>