karl-heinz blenk (Karl at blenk.deceiver.org) wrote:
: 1) I am looking for a marker to detect apoptosis in dorsal root ganglion
: cells using light microscopy.
: I con faintly remember having heard something about Acrydyl-orange.
: Does anybody has any experience with this substance or any other
: suggestions to offer?
Karl-Heinz,
I'm also sending you an e-mail about this, but for the benefit of everyone
else:
You didn't say the conditions of your experiments, but if you are planning
to fix the tissue, I'd recommend performing TUNEL or ISEL techniques for
in situ staining. TUNEL is a fairly simply procedure which relies upon
DNA fragmentation in apoptotic cells as a marker. TdT enzyme appends
base analogs to the 3OH end of DNA fragments. Next, you add a primary
antibody that tags the analogs.
It's a clever, simple technique that works pretty well (but is also fairly
expensive).
ISEL is a variant of TUNEL.
You can get a TUNEL stain kit, called APOPTAG, from Oncor (USA). Ask for
the Peroxidase kit, which is a light stain. Kit cost is $300, which allows
you to do up to 50 slides (but probably 40 is more realistic). You'll also
need to develop a positive control protocol, requiring a bit of fooling around
with times of exposure. I have a protocol if you're interested.
Jason Eriksen
