Help with 2 color TUNEL
gkremmid at mad.adelaide.edu.au
Wed Aug 7 17:49:32 EST 1996
At 22:48 8/6/96 +0000, Stacy Ferguson wrote:
>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,
What is a TUNEL assay?
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