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

[gayle callis]

Dear Merissa, 

Ray is correct about using serums with lectins.   In fact you really are NOT doing immunostaining unless you are using an antibody made to recognize that lectin e.g. anti-lectin therefore a normal serum block is not needed.    Serums in fact contain glycoproteins that bind to lectins, so BSA is a better protein carrier for buffers and diluents if you want to make up your own.  We used Jacksons immunoglobulin protease free BSA.   Ray gave good advice on blocking techniques.   However, if you do anti-Lectin, you would be doing IHC and then blocking is legal.   If you want a protocol for lectin IHC,  I will be happy to forward via private email.   With some lectins for direct staining,  you should use the Lectin buffer that has no phosphate ions, as explained by Vector technical services.   I also have a recipe for the Lectin buffer if you want it.   Avidin/biotin blocking is still needed with biotinylated lectin,  particularly is the tissue is known to have endogenous biotin.  Be sure to find out if your lectin is sensitive to phosphate ions, or use TBS or the Lectin buffer.  
 
Also, if you are doing a non-IHC direct lectin-biotin staining, you must do the correct negative control.  For SNA, this is an elution with 500 mM lactose in buffered saline followed by 500 mM lactose in acetic acid to finish elution.    Buffer alone is NOT a negative control.   For the lectins we worked with, we diluted the lectin (working concentration) in the recommended mM inhibition sugar and let it sit in the refrigerator overnight, warmed to RT just before use as negative control.   This allows the lectin to bind to its specific sugar, and not to glycoproteins in the tissue, but keeps the biotin, and in our case, fluorophore in the negative control.         

There is an excellent, inexpensive book, Lectin Histochemistry, a Concise Practical Handbook by SA Brooks, AJC Leathem and U Schumacher that tells all about using many lectins, protocols for lectin IHC and lectin direct binding staining.     An interesting side history is the founder of Vector is a lectin expert.    

For those doing IHC with anti-lectin,  antigen retrieval may be needed per James Watson reply and is included in the Lectin IHC protocol I have.    I am presuming he was doing true IHC for his lectin work.  

Gayle Callis
HTL/HT/MT(ASCP)

 



-----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 10:53 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
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 

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