Re: [Histonet] Request for closure

From:Todd Sherman

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Hi Rebekah,

The first "victim" of the guilt trip. :)

First, thanks for the follow-up. I know there are many that will review
your addendum (me included) to gain a better understanding of what works
and what doesn't.

Second, I tried to search the archives to review your previous post but
the search engine was not working, so I'll just fly by the seat of my
pants here. I've done a lot of work with eNOS in sheep lung (paraffin
and frozen) but it has been a while so some particulars seem a little
fuzzy. But, we used mouse monoclonal primaries from Transduction
Laboratories with great success in sheep. I don't remember the product
number off hand but if you do a search in this archive (when it comes
back online) for "eNOS sheep lung", I'm sure you'll find some of my
previous postings for the primary used. I believe Trans. Labs. was
acquired by BD Biosciences so a little review on your part might be
required. I found a link to what looks like the exact same mAb product
that we used so I'll list it for convenience. Again, please review their
website if the product, or this line of inquiry, looks interesting to you.

I know this doesn't address your blocking issue directly, but the eNOS
primary worked so well that you may be able to get by without going
through too many blocking hoops. Also, antibodies primed for westerns
often fail in IHC. They are designed to recognize a more linearized,
denatured peptide which doesn't always jive that well with the native,
three-dimentional conformation. Just a suggestion if you hadn't already
read this.

Hope this helps,

Todd Sherman
HistoSoft Corporation
"Biology in a new form..."
Member Services:


| ...As to the
| antibody not recognizing the sheep eNOS, I've been told that the mouse
| antibody I'm using has been used succesfully in Western blots with sheep
| tissue.

| Thank you all so much for your advice!
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