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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