[Histonet] Staining FFPE with biotinylated SNA - how to block?

[koellingr <@t> comcast.net]

Hi Merissa, 

don't know if you got any private idea responses so I'll throw in my opinion.  I would always worry about some of the things you are mentioning and that are standard thoughts regarding biotin block, retrieval, etc in IHC. 

But I would think about your serum, which I steadfastly avoided with SNA or any lectin I used.   Lectins look at glyco components and serum (or serum substitutes) can be full of glycoproteins and the target then is the blocking serum for your lectin which can cause bad background.  I did and would use washes, diluents, etc that had NO serum or milk or anything like that in them.  You can make your own, completely free of potentially having glycoproteins or Vector sells some.  For some lectins (look at a list of target sugars) you maybe can get by with serum or milk and such to block but many I've found you just can't. 


Ray (still in, whoever would have guessed, once again rainy Seattle) 

----- Original Message -----

From: "M.O." <modz9636 <@t> gmail.com> 
To: histonet <@t> lists.utsouthwestern.edu 
Sent: Tuesday, April 15, 2014 5:59:51 PM 
Subject: [Histonet] Staining FFPE with biotinylated SNA - how to block? 

Hello Histonet! 

I ran a trial on FFPE mouse samples with a biotinylated lectin, SNA from 
vector.  The SNA is Biotinylated Sambucus Nigra Lectin (Elderberry).  I 
have never stained with anything like this, so I ran a test. 

I deparaffinized, blocked with NGS, incubated overnight at 4C with the 
diluted biotinylated SNA.  On the second day, I used Vector's ABC kit and 
alkaline phosphatase (red) kit. 

Once stained, I noticed a lot of background.  After looking into the 
blocking step, would a biotin/avidin blocking step be the correct step 
instead of a serum because I don't have a secondary?  How do I know what 
needs to be blocked - biotin, avidin or both?  Is there a way to do this 
without a kit and use solutions I may have in my lab? 

Thank you for your help! 

Sincerely, 
Merissa 
_______________________________________________ 
Histonet mailing list 
Histonet <@t> lists.utsouthwestern.edu 
http://lists.utsouthwestern.edu/mailman/listinfo/histonet