[Histonet] Frozen Rat Tissue Staining

[Stacy Deppeler]

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.


-- 
Stacy Deppeler.  (Aussie Expat)

Research Assistant
Department of Ophthalmology
King Abdulaziz University Hospital
King Saud University
Riyadh, KSA