[Histonet] Fluorescent Apoptosis Detection

From:"Melissa Gonzalez"

Hi Wai Kam
My experience with the Roche kit has been pretty unsuccessful. Only because some lots work great and others don't. 
The reason you are seeing so many positive cells is that they manufacture the labeling enzyme pretty strong, and depending on the application/tissue, it can end up labeling all of your cells, instead of just some of them. Usually when I get a new kit, I dilute the TUNEL reaction mixture anywhere between 1:5-1:20 using their TUNEL dilution buffer. Use DNase as a positive control which each dilution. Regardless of the dilution, your positive should label ~100%. If it is less than 80% positive, you have diluted the reaction mixture too much. 
If you counterstain with DAPI, all your non-apoptic cells will have a blue nucleus. 
If you use the TMR kit, your apoptotic cells will have a red nucleus, and if you use the FITC kit, the positive cells will be green. 
Because of this hassle I now use an anti-Caspase 3 antibody which works great for detecting apoptosis. (from R&D systems)

Good luck,

Message: 5
Date: Thu, 19 May 2005 09:50:29 +0800
From: "Chan Wai Kam" 
Subject: RE: [Histonet] Double fluorescent stains for apoptosis
Cc: histonet@lists.utsouthwestern.edu
Content-Type: text/plain;	charset="windows-1250"

Hi Kelly,

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.  

Thanks again,
Wai Kam


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