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

[James Watson]

With our lectin staining on paraffin embedded tissue we use Avidin/Biotin blocking prior to antibody application.  Some protocols we do use HIER before the blocking.  One important item is that some lectins require a specific diluent.  The dilution can be as high as 1:12,000.  Lectins are well known for binding with multiple tissue entities, sometimes you can reduce the staining on unwanted tissue entities by reducing your incubation time or dilution.  We do not use serum blocking.

For PNA we use:
PNA Diluent
Calcium Chloride…………………..…………………  0.0111 gm.
Magnesium Chloride…………………………………  0.0203 gm.
Manganese Chloride…………………………………  0.0125 gm.  
Distilled Water……………………………………….….  100.0 ml

James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation
Tel    858-332-4647
Fax   858-812-1915
jwatson <@t> gnf.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of koellingr <@t> comcast.net
Sent: Wednesday, April 16, 2014 9:53 AM
To: M.O.
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Staining FFPE with biotinylated SNA - how to block?



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
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