[Histonet] Frozen Rat Tissue Staining
sdeppeler <@t> ksu.edu.sa
Sun Feb 13 13:24:19 CST 2011
The tissue was frozen in a slurry of isopentane and dry ice.
Department of Ophthalmology
King Abdulaziz University Hospital
On 13 February 2011 20:21, Montina Van Meter <Montina.VanMeter <@t> pbrc.edu>wrote:
> How are you preserving the tissue prior to sectioning (i.e. liquid
> nitrogen, isopentane, etc.)? Flash freezing is essential when dealing with
> unfixed tissue.
> Sent from my iPhone
> On Feb 13, 2011, at 3:10 AM, "Stacy Deppeler" <sdeppeler <@t> ksu.edu.sa>
> > 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
> > 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
> > quick summary:
> > 1. Cut 10um sections of rat eye at -20C, mount on plus slide and air dry
> > 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
> > 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
> > at a loss as everyone has different opinions on fixing and air drying.
> > tissue did appear to be intact after cutting and fixing when observed
> > the microscope.
> > Any input on my technique would be greatly appreciated as its hard to
> > 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
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