[Histonet] Re: Double fluorescent stains for apoptosis (Chan Wai Kam)

[Todd Sherman]

Hello Julee,

Since you are using a kit and someone selected it for TUNEL, maybe you  
could consult them first. Or, if that source is no longer available, try  
the vendor. They will have a technical staff to help you troubleshoot.  
I've done TUNEL staining but not with the kit you mention and not recently  
enough to provide dependable guidance. The vendor, or maybe manufacturer  
is more precise, earn their higher kit fees for such services so certainly  
query them for the subtleties of their product.

Regards,
Todd


Todd Sherman
President
HistoSoft Corporation

>>>>>>>>    www.histosoft.com     <<<<<<<<
<<<<<<<< Biology In A New Form (c)>>>>>>>>

On Wed, 18 May 2005 12:00:26 -0500,  
<histonet-request <@t> lists.utsouthwestern.edu> wrote:

>
> Today's Topics:
>
>    9. Double fluorescent stains for apoptosis (Chan Wai Kam)
>   10. Re: Double fluorescent stains for apoptosis (John Kiernan)
>
> ----------------------------------------------------------------------
>
Message: 9
Date: Wed, 18 May 2005 11:28:34 +0800
From: "Chan Wai Kam" <doscwk <@t> nus.edu.sg>
Subject: [Histonet] Double fluorescent stains for apoptosis
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<E50AB2A0A689B54C8B61CB6E561A82B8D3692A <@t> MBOX02.stf.nus.edu.sg>
Content-Type: text/plain;charset="windows-1250"

Hi,

We?re doing a study on the effect of compression and degeneration of  
rabbit intervertebral discs, and we're using the In
Situ Cell Death Detection Kit, TMR red (Roche) for the detection of  
apoptosis by fluorescent microscopy.  As our lab is
new to this technique, we would be grateful for any advice on the correct  
apoptosis stain/kits to use.

Our problem in using this kit is that we get a large number of positive  
stained cells (all red).  As we would like to
find out the percentage of apoptotic cells, we need a protocol which can  
give us a double stain which according to one of
the papers is bright green (tunnel positive) and light red (unaffected  
cells).

Any advice and protocols would be greatly appreciated.

Thanks in advance
Julee Chan
Orthopaedic Surgery
Histology Lab
National University of Singapore
------------------------------

Message: 10
Date: Wed, 18 May 2005 00:54:09 -0400
From: John Kiernan <jkiernan <@t> uwo.ca>
Subject: Re: [Histonet] Double fluorescent stains for apoptosis
To: Chan Wai Kam <doscwk <@t> nus.edu.sg>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <428ACA71.6CE8BF2B <@t> uwo.ca>
Content-Type: text/plain; charset=iso-8859-1

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
cell death.

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
too".
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
    kiernan[AT]uwo.ca
    http://publish.uwo.ca/~jkiernan/
    http://instruct.uwo.ca/anatomy/530/index.htm
------------------------------