[Histonet] RE: HRP-conjugated secondary and biotinylated-secondary
Dear van der Loos
Thank you very much for your reply with historical overview.
Thank you everybody who replyed to my question.
--------- Mensagem Original --------
From: "C.M. van der Loos"
Subject: RE: HRP-conjugated secondary and biotinylated-secondary
Date: 31/01/05 14:29
The existence of both biotinylated- and HRP-conjuated secondary
antibodies reflects the development of IHC techniques into
more staining efficiency/sensitivity. In the old days, a 2-step
staining method using a HRP-conjugated secondary anti-mouse or
anti-rabbit was common practice. Mid 80-ies the 3-step staining method
using a biotinylated 2nd step reagent and streptavidin/HRP conjugate
or streptavidin-biotinylated HRP complex became very popular because
of its superior staining efficiency/sensitivity. The disadvantage here
is the occurrence of endogenous biotin in many tissues. Endogenous
biotin is not present in untreated formalin-fixed and
paraffin-embedded tissue sections, but will be retrieved especially
when using Tris-EDTA pH8-9.
Only recently we have gone to a 2-step staining procedure again, now
based on both secondary antibody and HRP attached to a polymer
backbone. The efficiency/sensitivity of these polymers is comparable
with the 3-step streptavidin/HRP procedure, but avoids the problem of
I hope this small historical overview helps.
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
fax: +31 20 6960389
----- Original Message -----
Date Sun, 30 Jan 2005 0:18:56 +0600
Subject [Histonet] HRP-conjugated secondary and
Is it a common practice to use HRP-conjugated secondary antibody
instead of biotinylated secondary antibody for immunohistochemistry
(with DAB developing system)? I am confused about the advantage/
disadvantage of using HRP-conjugated secondary antibody for
UNICAMP, SP, Brazil.
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