Methanol fixation for primary cultures neurons.

[nikhil pandya]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Greetings!
A bit of Neuroscience specific question.

I have been testing antibodies for Post synaptic density proteins like
PSD95 and AMPA and NMDA receptor in primary hippocampal neurons. I am
finding it difficult to get good labelling in the PSD. Is it recommended to
use methanol to fix and stain PSD proteins in general? Are there any
disadvantages to using methanol instead of PFA fixation?

--
Nikhil Janak Pandya.
E mail: [hidden email]
Contact no: [phone]

[Tanneke den Blaauwen]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Greetings,

I do not have experience with neurones, but bacteria shrink considerably if you fix them with methanol and all proteins are in principle denatured because of dehydration. PFA is much milder. Both methods give an osmotic shock, which might displace some proteins. In general in bacteria, the proteins are only displaced if they are freely floating on the exterior of the cytoplasmic membrane.

Kind regards,

Tanneke den Blaauwen

On 22 Oct 2014, at 21:42, nikhil pandya <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Greetings!
> A bit of Neuroscience specific question.
>
> I have been testing antibodies for Post synaptic density proteins like
> PSD95 and AMPA and NMDA receptor in primary hippocampal neurons. I am
> finding it difficult to get good labelling in the PSD. Is it recommended to
> use methanol to fix and stain PSD proteins in general? Are there any
> disadvantages to using methanol instead of PFA fixation?
>
> --
> Nikhil Janak Pandya.
> E mail: [hidden email]
> Contact no: [phone]

Tanneke den Blaauwen
Assoc prof Bacterial Cell Biology
SILS, UvA
Science Park 904, C3.106
1098 XH Amsterdam, NL
[hidden email]

[Daniel Gitler]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Nikhil,
First - I like your email address...
Out of the list you ask about, I have used just PSD95 antibodies. They worked very well in samples fixed with methanol, but did not work whatsoever in PFA fixed samples. So, at least in my case, this was a necessity. At the light-microscope level I could not see any obvious problem with the methanol fixed sample, when comparing other markers I had used (like presynaptic proteins) in methanol and PFA fixed samples. Notice that when hydrating the cultures after the methanol fixation step, the solutions bubble quite a lot. Still, the end results were as expected, and quite nice to behold Hope this helps.
Daniel

Daniel Gitler, Senior Lecturer
Department of Physiology and Cell Biology
Faculty of Health Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of nikhil pandya [[hidden email]]
Sent: Wednesday, October 22, 2014 10:42 PM
To: [hidden email]
Subject: Methanol fixation for primary cultures neurons.

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Greetings!
A bit of Neuroscience specific question.

I have been testing antibodies for Post synaptic density proteins like
PSD95 and AMPA and NMDA receptor in primary hippocampal neurons. I am
finding it difficult to get good labelling in the PSD. Is it recommended to
use methanol to fix and stain PSD proteins in general? Are there any
disadvantages to using methanol instead of PFA fixation?

--
Nikhil Janak Pandya.
E mail: [hidden email]
Contact no: [phone]