[Histonet] RE: weak antibody signal
I have three detection/visualization procedures for you to consider
for testing (no. 3 is based on the assumption that you are intended to
stain FFPE sections; no. 1/2 can be used for cryo's too):
1. Most simple is just to repeat what you did so far but ending with
Vector NovaRed kit instead of DAB. This very sensitive kit has
helped us in similar situations a couple of times here.
2. We have been pretty successful lately with the detection of goat
antibodies using a 2-step method ending with a polymer. The second
step was a rabbit anti-goat Ig (Dako) 1:5000, 30 min RT and the
final step EnVision+ anti-rabbit. DAB+ was used for chromogen.
3. As you said the CSA kit might be a good solution too. In case of a
goat antibody your second step should be rabbit anti-goat Ig/HRP
(Dako) 1:100, 15 min, RT. Then continue with Amplification
reagent, etc. Please realize that the reaction product after
tyramide amplification is somewhat more diffusely localized than
with other IHC detection procedures.
In all three cases you should perform new dilution series of your
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
Date: Tue, 22 Nov 2005 10:44:27 -0600
From: "Till, Renee"
Subject: [Histonet] weak antibody signal
We have a BTEB1 goat polyclonal antibody that is staining very weakly
using a Vectastain kit and DAB. I personally like to order my
secondaries and streptavidin separate, that way I can get F(ab')2
fragements or what have you, if I want. I was going to suggest trying
this, as it is has worked better for me on some stains than the Vector
kits. Is there anything else that could be done to boost the signal? I
had thought of CSA kits and the like, but from what I can tell the
kit at least isn't for goat. Would it work better perhaps using
fluorescence instead of DAB?
Renee' Till, HT
Arkansas Childrens Nutrition Center
Little Rock, AR
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