Re: [Histonet] one heck of a IHC question regarding fixedfree-floating brain sections

If you want to inactivate the residual hrp just incubate in hydrogen peroxiede step again. I do sequential double stains with two HRP based chromagen/substrates and do this all the time. I use 0.3% or 3% but I don't work with thick sections so you may need to tweak that a bit. I agree just redo the protocol. Using a different color might be a good idea though just so you can track any interference from your first stain.

-----Original Message-----
From: Linke_Noelle 

Date: Mon, 19 May 2008 16:13:22 
To:"Mejia, Maria"
Subject: RE: [Histonet] one heck of a IHC question regarding fixed
	free-floating brain sections

Hi Maria,

I say go back with the right antibody(don't use a goat Ab.... use rabbit or mouse), followed by anti-whatever(rabbit or mouse), and you'll need to use a Alk-phos detection and chromogen so you don't light up any of the HRP labeled stuff.

Or....if you know they will be negative, I would think that doing the DAB development would simply bind up the HRP sites (if any are there), then you could start your staining all over....


Allergan, Inc
2525 Dupont Drive RD-2A
Irvine, CA 92612

-----Original Message-----
From: [] On Behalf Of Mejia, Maria
Sent: Monday, May 19, 2008 3:52 PM
Subject: [Histonet] one heck of a IHC question regarding fixed free-floating brain sections

Dear All,

I have one heck of a question for everyone & especially those who work with fixed 40um
free-floating brain sections. A number of our very precious brain sections were mistakenly
stained with the (wrong) primary (anti-human NTN) antibody. 

Now, we KNOW that there is NO NTN (which influences a variety of neuronal populations in 
the brain) in these sections. All sections (except 1) have NOT gone through the DAB 

So, we need to know [if] & [how] we can restain these sections with the correct primary 
antibod Here the protocol using the wrong primary antibody - please read carefully.

-wash sections in PBS x3 - 5 mins ea.
-block in 1% H202/PBS - 20 mins
-wash sections in PBS x3 - 5 mins
-block in casein biocare sniper - 30 mins
-incubate in anti-goat primary NTN 1:4000 antibody in diluent - stained overnight.
Next Day:
-wash in PBS x3 - 5 mins ea.
-incubate in Biocare Medical Goat probe - 1 hr.
-wash in PBS x3 - 5 mins ea.
-incubate in Biocare Medical Goat HRP - 1 hr.
-wash x3 PBS - 5 mins ea.
-develop ONLY ONE SECTION in Vector DAB - here's when we caught our mistake.
Since we only developed 1 section in DAB - the other sections are still sitting in PBS @ 4C &
have NOT gone through the DAB development.

My question is can we & how - do we re-stain these undeveloped (no DAB) sections using the 
correct primary antibody either polyclonal or monoclonal using either the same
goat probe & goat HRP or another polymer combination. Then develop the resulting end
with DAB??????? If we can how do we do it????????

Please any insightful suggestions & tips will be greatly helpful to us. Please Dr. Loos let me hear
from you!!!


Maria Mejia
Histology Manager
Department of Neurosurgery
San Francisco, CA 94103

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