Help with 2 color TUNEL

Stacy Ferguson sef at med.unc.edu
Tue Aug 6 17:48:36 EST 1996


I've recently started using TUNEL assays to examine apoptosis of 
B cells. I've had no problem doing this. However, I'm having some
trouble with subsequent stains of the same sections using anti-
allotype specific antibodies and am hoping that someone here may 
have some advice.

Briefly, I'm working with a strain of mouse carrying a Igh transgene
of the a allotype. The allelic exclusion is leaky and endogenous 
rearrangements also occur. I'd like to identify those B cells of each
allotype that are undergoing apoptosis.

So far I've tried two strategies that haven't worked. Individually, I have
no problem staining with the allotype specific antibodies alone or 
performing the TUNEL assays alone. First, I tried fixing the sections 
in formalin, post-fixing in EtOH/HOAc, performing the TUNEL assays and
then going through my anti-allotype staining procedure. This failed. I
have also tried performing the TUNEL assays through the TdT reaction and
simultaneously staining with both the anti-digoxigenin antibodies and
the allotype reagents. The substrate reactions were done independently 
using DAB (which worked fine), followed by the alk.phos. reaction using the
Vector Labs "Vector Blue" system. The blue staining was completely absent.

I have also tried doing the TUNEL assay alone until completion, followed
by incubating the sections with anti-allo reagents. This also failed (the 
TUNEL assay, again, worked fine).

In the next few days, I'll be trying the following and would appreciate
any hints regarding my concerns:

1. Fix the sections with formalin, perform the anti-allo reactions and
   develop them with BCIP/NBT. Postfix with the EtOH/HOAc and perform the
   TUNEL assay.

   My questions here are: Is the BCIP/NBT product stable through the 
   EtOH/HOAc step? WIll there be a problem with getting my TUNEL assays 
   to work after all that manipulation?

2. Perform both fixation procedures in sequence, do the anti-allo
   reactions first, followed by the TUNEL assays. Is there anything 
   in the Alk. Phos. substrate reaction that may later inhibit the 
   TUNEL reactions?

I realize that these questions are rather specific, but since I've only
started doing TUNEL assays in the last few weeks, any suggestions for 
alternative staining procedures in addition to the two I've tried and 
the two I intend to try this week would be appreciated.

Thanks in advance,


Stacy





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