[Histonet] lectin histochemistry questions

[koellingr <@t> comcast.net]

Li Fang,

In addition to some of the other comments, do not forget the role of the buffer in lectin histochemistry.  Do not know which lectins you are using on your prostate study but some lectins are problematic in their need for divalent cations (calcium or magnesium) in buffers for binding to the sugar residue.  Some lectins are no problem and can use PBS but some are sensitive and some (I think those targeting mannose?) absolutely require divalent cations.  Since you can't be putting such stuff in PBS without precipitating out the phosphate salt, a lot of people use Hepes buffers.  See the Vector website which in my opinion, although I have absolutely no connection with Vector, is the premier place to go for information on lectin histochemistry staining.

Ray Koelling
PhenoPath Labs
Seattle, WA
 
 -------------- Original message ----------------------
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
> Li Fang:
> As per your questions:
> 1- use acetone
> 2- if they are FS you do not need to dehydrate, you need to place them in a 
> buffer (PBS is advisable)
> 3- you do not need to block, remember that your lectin is the one that is going 
> to be "added" to the tissue for a later detection I cannot advise on the 
> remaining questions
> René J.
> 
> --- On Tue, 7/1/08, Yang, Li Fang <yangli <@t> EVMS.EDU> wrote:
> 
> From: Yang, Li Fang <yangli <@t> EVMS.EDU>
> Subject: [Histonet] lectin histochemistry questions
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Tuesday, July 1, 2008, 2:03 PM
> 
> Dear all,
>  
> I am a newer to lectin histochemistry. I need to work up a portocol using a
> panel of lectins for human prostate cancer tissue sections. I was advised to
> use fresh snap-frozen tissue, and fluorence conjugated lectin as a start point.
> Now I have 7 FITC conjugated lectins and frozen sections. But I still have some
> questions to ask for your help.
> 1. What is the suitable fixtive for the fresh tissue? 75%acetone? 25%ethonal?
> or ?%methonal?
> 2. Do I need to dehydrated sections? before or after fixation?
> 3. Do I need to block endogenous peroxidase if I don't use peroxidase-based
> detection?
> 4. Is it necessary to use some blocking reagents such as BSA in the staining
> buffer (lectin buffer as suggested in this website)? I tried 2% FBS/PBS and
> 1%BSA/PBS in the lectin staining by flow cytometry. They both saturated the
> binding of lectins, almost to the same extent. FBS contains a lot glycan
> residures, but how to explain the effect of BSA? it is even not a glycoprotein.
> 5. It is said that FITC may conflict with some autofluorescence in FFPE
> sections. How about in fresh sections? how to figure it out? 
> 6. How to evaluate the staining intensity? Is there a specific software
> required?
> 7. For lectin specificity, I plan to use inhibitory sugar. anybody know the
> solvents for lactose and N-acetylneraminic acid? I try to make 1M stock
> solution. They seemed not dissolved in water.
>  
> Sorry for so many questions. Any tips and protocol sharing would be greatly
> appreciated.
>  
> 
> 
> ::
> _______________________________________________
> 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