Re: GFAP and NeuN on mouse tissue?

From:Geoff McAuliffe

Hi Melissa:

    DAKO's rabbit anti-cow GFAP is outstanding. Lasts for years in the refrigerator. I have used it on mouse and rat CNS with beautiful results. On FFPE tissue I use a 1:6000 dilution with a Vector ABC Elite kit but I run the primary Ab overnight and use
nickel+cobalt intensification. On free-floating, fixed frozen sections cut at 20 microns on a sliding microtome the dilution is 1:60,000 (same kit, same intensification, no that dilution is not a typo). Thicker sections, 20-60 microns, are very nice for
astrocytes, you will see much more of the 3 dimensional character of the cells. Paraffin sections that thick are hard to cut, so fix, cryoprotect with sucrose, snap freeze and cut on a cryostat or a sliding microtome. GFAP survives fixation well. The few
times I have done fluorescence I used lower dilutions, about 1:6000 I think, again with Vector reagents. Keep in mind that not all astrocytes are GFAP-positive!
    I have no experience with the anti-NeuN. Go to the PubMed site and look for a recent paper on the subject. Or ask Chemicon, they will have some references. Most of the commercial suppliers keep track of publications with their products, for obvious

Melissa Gonzalez wrote:

> Hello,
> Does anyone have any experience using Chemicon's mouse anti-NeuN and/or DAKO's rabbit anti cow GFAP?
> I have seen a few discussions relating to FFPE human tissue, but we are working on mouse frozens for fluorescence microscopy..which leads into my next question, How thick can I keep (or how thin should I cut) the tissue (brain) for optimal visualization?
> Thanks in advance for your help!

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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