[Histonet] Re: Histonet Digest, Vol 87, Issue 24

[Amos Brooks]

Hi Stacy,
     I have done this on FFPE samples without terrible difficulty. I'm not
sure if the I did procedure will work well on frozen tissue, but I don't see
why it wouldn't. I also used HRP/DAB to visualize the reaction however a
fluorescent tag should be fine. I used an antibody from AbCam (ab49873) at
1:200 with citrate buffer (pH6.0) retrieval. It might not be working for you
now, but I'm sure you'll get it.

Good luck,
Amos

On Sun, Feb 13, 2011 at 1:00 PM,
<histonet-request <@t> lists.utsouthwestern.edu>wrote:

> Message: 3
> Date: Sun, 13 Feb 2011 12:03:25 +0300
> From: Stacy Deppeler <sdeppeler <@t> ksu.edu.sa>
> Subject: [Histonet] Frozen Rat Tissue Staining
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
>        <AANLkTi=JeTnjeEg=jwEt8XwmoybbSeisQDLYhJbXy5Ht <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi Histonetters,
>
> I've been trying to perform an immunofluorescent stain for Glutamine
> Synthetase on frozen rat eye sections and having very little luck. I put
> this down mostly to my lack of experience with frozen sections so I'm
> hoping
> I can find a little help here to guide me on where I am going horribly
> wrong. The problem I am having is that the retinal tissue is completely
> degraded by the time I am finished the stain.
>
> I have been using a staining protocol my PI gave me that his previous
> assistant used with good results, but for me is giving me nothing. Here's a
> quick summary:
>
> 1. Cut 10um sections of rat eye at -20C, mount on plus slide and air dry
> for
> 30 min.
> 2. Fix in -20C acetone for 10 min, air dry for 10 min.
> 3. 3X PBS-Tween20, 5 min.
> 4. 5% serum block, 10 min.
> 5. Primary antibody incubation, overnight at 4C.
> 6. 3X PBS-Tween20, 5 min.
> 7. Fluorophore-conjugated secondary antibody (Texas Red), 1hr.
> 8. 3X PBS-Tween20, 5 min.
> 9. IF aqueous mount with DAPI.
>
> I'm finding that the retinal tissue has already broken down significantly
> when I take the slides out from the overnight primary antibody incubation.
> My first thought is that the tissue is not fixed well enough but all my
> research so far tells me it should be. However, the more I read the more
> I'm
> at a loss as everyone has different opinions on fixing and air drying. The
> tissue did appear to be intact after cutting and fixing when observed under
> the microscope.
>
> Any input on my technique would be greatly appreciated as its hard to work
> out what you're doing wrong when you don't know what you're doing right!
>
> Thanks.
>