Carol,

Ralph is right in suggesting to use biotin, because it is certainly a more
routine procedure to label proteins. However, DIG labeling reagents can be
easily purchased at Roche (no affiliation) and their procedures are usually
pretty straightforward and very reproducible. From my experience with DIG,
botin and FITC in in situ hybridization I can say that the most sensitive
and least likely to produce background is clearly DIG. In in situ
procedures you need maximal sensitivity as compared to ICC, and with biotin
we always have problems with endogenous binding activities for
avidin/streptavidin, which are rather hard to block. We have similar
problems with FITC, probably due to non-specific binding to tissue.

On the other hand, we never encountered endogenous DIG reactivity, even
with maximum sensitivity detection systems (AlkPhos with NBT/BCIP detected
for 3-4 days). Thus my advice would be, if you expect to need high
sensitivity, go with DIG. All anti-DIG antibodies/conjugate from Roche are
very good. If you do not expect to need high sensitivity, go with biotin,
because you most probably already have all secondary reagents you need in
your lab.

Paul Klosen

>Carol-
>
>I would suggest biotinylating your antibody since it will allow you to
>amplify your reaction using streptavidin etc.  I think Sigma sells a
>biotinylkating reagent called NHS-long chain biotin.  It is fairly easy to
>put the biotin on the antibody and get rid of any unreacted biotin.
>
>Ralph Marcucio, PhD
>
>-----Original Message-----
>From: Carol Chang [mailto:carolc50@hotmail.com]
>Sent: Wednesday, November 14, 2001 10:50 AM
>To: histonet@pathology.swmed.edu
>Subject: FITC or DIG?
>
>
>Hello:
>I need to use a human antibody on human tissue.  So I am going to label the
>antibody either with DIG or with FITC. Could anybody tell me which one is
>better? And which anti-DIG or anti-FITC antibody works best? The histonet is
>the best. It helped me a lot.
>Thanks in advance.
>Carol

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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur
12 rue de l'Universite
F-67000 Strasbourg, FRANCE
tel: 03.90.24.05.01    fax: 03.90.24.05.28
===============klosen@neurochem.u-strasbg.fr==============================