Re: Tyrosine Hydroxylase

From:Geoff McAuliffe <mcauliff@UMDNJ.EDU>

Anila Syed wrote:

> Dear All,
> Sorry if this has been asked before. I have a protocol for Tyrosine
> hydroxylase which I am using to stain 50um brain stem sections. It has a 4
> day incubation of primary on it at 4 deg C and I was wondering whether
> anyone has had any experience using this ab. My question is, can I do a
> shorter incubation at room temp and get the same results?

    When I did a lot of TH on mouse and rat brain (20 micron frozen sections,
free-floating, or 10 micron cryostat sections on slides) I stained with primary
overnight at room temp., followed by an ABC Elite kit. Our fixation times were
short, 4 hours in fresh 4% paraformaldehyde. We also got this to work on
tissues rich in TH (adrenal medulla) embedded in paraffin.
    There are a lot of variables to think about. Triton in to enhance
pentration? Frozen versus vibratome sections, time in fixative, etc. Your best
bet is to do your own experiment. Run your usual proceedure and pull sections
from the primary after one, two, three and four days. See a difference?

> A secondary question I have is how reliable is immunostaining for tissue in
> which you want to count and measure cells? Does one get consistent results
> with a given ab and a given protocol? i.e. if one used the same protocol on
> the same tissue section, would the same cells always be stained and to the
> same extent?

It should be very consistant IF all steps in the proceedure are kept
consistant. Again, this must be determined by you for your material and

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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