Re: [Histonet] Tunel and IHC

From:"Thomas Pier"

Renee,
I've had great results with double labeling using Roche's TUNEL w/
flourescein kit.  I just follow the directions using the permeablization
solution made with sodium citrate and triton x100.  Since this TUNEL kit
doesn't actually use an antibody, it is very easy to use for double
labels.  I actually did some tripples with it if you include DAPI on my
frozens.  I've done this with FFPE and fresh frozen tissues with good
results.  I would imagine that it should work fine on cells too.

Tom Pier
TRIP Lab
Department of Pathology
UW School of Medicine and Public Health

>>> "Till, Renee"  06/20/06 9:58 AM >>>
Has anyone tried to co-localize an IHC and Tunel on cells with
fluorescence? I saw tons of messages about Tunel, but nothing about this
specifically. It seems to me that in some ways cells would present more
of a problem. Another tech (I don't do just cells) is trying to do Tunel
and Pten fluorescence on the same slide. He has tried doing the Tunel
first and then the Pten, and it seemed to work. He is going to try doing
it the other way also to compare. You could do multiple slides, but
would it be the same as if you were doing tissues that you could use
nearly identical sections for each of the stains and the double?  What
I'm wondering is what are the problems that occur and how does doing
fluorescence factor into it. He mentioned not doing the proteinase K
step of the Tunel (because it would interfere with the subsequent Pten
staining) and still got staining. Is that valid?  To just skip a step
that you would normally do? 

He checks the first stain before starting the second, but doesn't take
any photos until both are done. Shouldn't you at least take a photo when
checking the first stain to see if the second affects it any? Just seems
like he has too many variables.

 

Renee' Till, HT

Research Assistant

Arkansas Children's Nutrition Center

1212 Marshall St./N2021

Little Rock, AR 72002

Office (501)364-2785

Fax (501)364-3161

 


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