[Histonet] Double fluorescent stains for apoptosis

[John Kiernan]

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
_______________________________
Chan Wai Kam wrote:
> 
> 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
>