Re: [Histonet] lectin histochemistry questions

From:Rene J Buesa



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  wrote:

From: Yang, Li Fang 
Subject: [Histonet] lectin histochemistry questions
To: histonet@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.
 


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