GC-IB4 staining problems

Hi everyone, I recently bought the fluorescently conjugated Isolectin GS-IB4 (Alexa Fluor 568). It doesn’t come with any information how to use it. Can you tell me how you guys dilute it to stain cells? Thanks in advance for your help.

Can you provide a catalog number? If this is the one I’m thinking about (Life Tech), I diluted it as indicated on the data sheet, and then I use at 1:1000 dilution during 2ndary staining for 1 hour at RT.

I used PBS w/ CaCl2 to dilute to 1 mg/ml.

Here is the manual: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp21410.pdf

the catalog number is I21412 from thermo fisher scientific. I normally incubate my secondary antibodies in blocking buffer, is that what you are doing too?

Yes. It’s pretty straight forward. Just stick in the iB4 with your secondary antibodies.

We incubate the GS-IB4, Alexa-647 (1/500, Thermo, I32450) with the first antibody overnight. Incubation with the secondary antibodies also works okay, but the incubation with the first antibody results in a much better staining, at least in our hand.
IB4-647

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Nice @tberta. I remember you taught me to do in the secondary. But it looks lke now you prefer overnight. I guess it’s time to switch.

thanks guys these are all very helpful

Hello!
could you tell me what concentration between 0.1 and 1mM of CaCl2 did you use to dissolve IB4? It was in water?
Thank you :slight_smile:

I’m sorry. I don’t recall. I’m not even sure if Ca was needed. I would just follow the manufacturer’s instructions.

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Has anyone tried using the IB4 stain without combining it with any other primary/secondary antibodies? I have been having trouble with severely high non-specific binding when using it alone at different conc. or even incubating for different durations where I get a lot of fluorescence from what looks like extracellular matrix. Could it possibly be that combining it with another primary/secondary antibody helps bloth this non-specific binding?

I am using Thermo I32450 on DRG tissue sections.

What is the diluent when it’s alone? Blocking buffer of some kind? PBS?
Have you tried a different lot or brand of IB4?