RE: more info again, End PO block

From:Chris van der Loos

Dear Becky,
There is a new endogenous blocking solution from Dako called "Dual 
endogenous enzyme block" (S2003). It will kill both endogenous peroxidase 
and alkaline phosphatase activities in one 10 min incubation step prior to 
your immunosteps. As this stuff is new my experience is only limited. So 
far it seems very effective without damaging the tissue epitopes.
Furthermore, as Gayle Callis already wrote, methanol + 0.3% peroxide (20 
min, RT) is very effective to block endogenous peroxidase activity in FFPE 
sections. Because those sections have been trough alcohols several times 
during embedding and dewaxing, the methanol has no additional damaging 
effect. Be aware that methanol can be very damaging to many tissue epitopes 
when immunostaining cryostat sections!
I have also tried to prolong the incubation time of a lesser effective 
endogenous peroxidase blocking method as you wrote in your first mail, but 
it didn't help.

Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands

At 11:47 AM 8/1/2003 -0400, you wrote:
 >I'm sorry about not giving enough information.  I have had several
 >responses asking for more details.  So, I'll try again!
 >I am performing IHC staining.  I am using a DAKO autostainer with
 >the LSAB detection system.  I use DAB for my chromagen.  My negative
 >mouse and negative rabbit serum slides have quite specific staining
 >in the granlocytic series of WBC's(No matter what pretreatment -
 >I apply no pretreatment, Proteinase K, or use Microwave at pH 6 and
 >10).  Microwave pretreatment is the worst, but I get the staining
 >most of the time.  Polysegmented neutrophils seem to be the main
 >culprit.  Most of the times my Docs can read through this, but they
 >are requesting my experimentation to find a "cure" for the situation,
 >so they can honestly say, "no staining was seen in the negative
 >control". One of the suggestions I have received is to do an Avidin/Biotin
 >Block. I will give this a try, but if there are any other ideas out
 >there:  DON'T hold back!  THANKS AGAIN! 

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