[Histonet] Double fluorescent stains for apoptosis

[Swaram]

Dear Chan,

I wont try to evangelize or advise you on the benefits of using the
library. But here is something you can do for TUNEL Roche Kit. I use one
with fluorescein. But if you read the product inser that came with it,
you will find something called TUNEL Dilution buffer. Use that to dilute
your enzyme mixture in serial dilutions of 1:10 to 1:100 and so on. You
will see that the background gradually fades away. Standardize it for
your satisfaction. Also make sure that you have a good Block for the
slides. Otherwise you will find the Fluorescein all over the place.
http://www.roche-applied-science.com/pack-insert/1966006a.pdf

Should work...!

Best Wishes,
Swaram

And here is a previous reply which might be slightly more informative on
the same subject

Dear Toshi,
TUNEL is not a waste of time, just don't use the Roche kit! I had the
same problems with it: lots of normal looking nuclei staining positive.

Now I am using the Trevigen TACS kit (in Canada sold through
BIO/CAN)and it works great. You can buy individual components, no need
to get the whole kit each time you run out of a component. I buy the
TdT enzyme, biotinylated dNTP nucleotides, Co 2+ cations, Proteinase K,
TdT Labeling buffer and TdT Stop buffer. I follow their protocol up to
the Stop buffer step, then I vizualize the incorporated biotinylated
nucleotides either with my usual IHC Streptavidin-peroxidase, Nova-Red
incubations, or for fluorescence, with Streptavidin-Alexa from
Molecular Probes.

The staining is very clean with nuclei of apoptotic morphology staining
positive. You can check my double immunofluorescence for TUNEL and
cleaved caspase-3 in mouse thymus in our recent paper: Circulation
2005;111:1814-1821, Supplemental on-line Figure 5. TUNEL specifically
stained nuclei in cells that had cytoplasm positive for cleaved
caspase-3.


Sarka Lhotak
McMaster University, Hamilton, Ontario





\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 
>
>  
>