RE: [Histonet] Double fluorescent stains for apoptosis
Thanks for your understanding. Frankly I did feel chastened and thought I'd better go and do my homework more thoroughly. It's true that I had hoped that some kind souls in histoland could offer me their advice on the correct protocols or kits to use as the kits are so costly and I'm wandering into unfamiliar territory.
I'm not a clinical but lab staff in research and my work has been mostly on routine histology of decalcified tissues. I've no prior experience in apoptosis and so find it a bit confusing with all the protocols I've found on the web. The PI whose project I'm assisting isn't of much help either as he was on a short attachment with our dept, finished the experimental surgery part and left the bulk of the work for the lab staff to complete. He's happily in some other country and would drop an email every so often expecting results. So frankly I'm unsupervised and trying to do the job as best as I can.
The Roche kit I used (In Situ Cell Death Detection Kit, TMR red for fluorescent microscopy analysis) comes with a protocol but the results do not seem accurate as there were too many cells stained positive. I also realized too late that I should be going for something that shows up the unaffected cells as well since we need to get a percentage. So back to the web I went to do more searches but still not any wiser. And that's when I thought about all those experts out there in histoland who could perhaps point me in the right direction.
From: email@example.com [mailto:firstname.lastname@example.org]
Sent: Wednesday, May 18, 2005 9:21 PM
To: John Kiernan
Cc: Chan Wai Kam; email@example.com
Subject: Re: [Histonet] Double fluorescent stains for apoptosis
The reason people subscribe to histonet is to reduce time spent "going
to the library and spending half a week immersing yourself in the
published literature of cell death and the methods used to recognize how
it happens." This is achieved by asking your peers and "experts" for
help. You should copy and send this response to everyone asking advice
when they can simply take a week and look it up. I know I don't have
that kind of time, and it is clear that Chan Wai Kam does not either.
Chan Wai Kam: I would help you, my friend, but I do not have experience
with the Roche kit.
John Kiernan wrote:
>Dear Chan Wai Kam,
>Your enquiry clearly indicates that you don't know how
>your bought kit works. The TUNEL (not tunnel! It's an
>acronym for Terminal Uridine Nick-End Labelling) family
>of methods has many shortcomings, all thoroughly
>documented in peer-reviewed papers that are cited in
>textbooks. TUNEL, intelligently used, has its place,
>alongside other techniques that indicate modes of
>Your "we need a protocol" plea is a way of saying
>"Tell me exactly what to do" instead of going to the
>library and spending half a week immersing yourself
>in the published literature of cell death and the
>methods used to recognize how it happens.
>Are you a clinical resident required to do a research
>project in a few weeks? This is the impression that
>I get from your email. The only sensible reply is
>"Go to the library and tell your boss to go there
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