[Histonet] one heck of a IHC question regarding fixed free-floating brain sections

From:"Mejia, Maria"

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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