RE: [Histonet] Endogenous Peroxide Treatment -IHC

From:"Tony Henwood"

>>"Why in TMAs always we found a liver as a control sample, which gives
us false result???????????????">>

Probably because it does.

If using the ABC demonstration method, I would always include a small
sample of liver in my negative control to gauge the technique's ability
to block endogenous biotin. And would you believe that the amount of
biotin stained would vary from batch to batch (why??- maybe slide drying
temp/time OR efficiency of antigen retrieval OR age of biotinylates
antibody OR age of blocking solutions OR ....)

For this reason we now use polymer technology.


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145 

-----Original Message-----
[] On Behalf Of
Margaryan, Naira
Sent: Tuesday, 16 September 2008 6:14 AM
Cc: Marilyn Johnson
Subject: [Histonet] Endogenous Peroxide Treatment -IHC

Hi histonetters,

I fully agree with Isaac.

We all know that the liver always gives a background even if 4 steps of
blocking were used (H2O2, Avidin, Biotin, Protein Block).

Why in TMAs always we found a liver as a control sample, which gives us
false result???????????????


Message: 2
Date: Sat, 15 Mar 2008 11:29:08 -0700
From: "Isaac Milos" 
Subject: RE: [Histonet] Endogenous Peroxide Treatment -
To: "Marilyn Johnson" ,
Content-Type: text/plain;	charset="us-ascii"

Hi Marilyn,

If you are running your IHC stains with an HRP detection kit with a
biotinylated secondary antibody, endogenous biotin within the tissue can
cause background staining to occur.  Often times, you'll see endogenous
biotin in a tissue that is particularly bloody, such as liver, kidney,
or brain.

You can use an avidin/biotin block before secondary antibody is applied
to account for this.  You can find this block through many different
companies - mine (Cell Marque) offers a good version.  The part number
is CMX222 and it can be ordered at 1-800-665-7284.

Happy staining!


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