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.
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
could you tell me what concentration between 0.1 and 1mM of CaCl2 did you use to dissolve IB4? It was in water?
I’m sorry. I don’t recall. I’m not even sure if Ca was needed. I would just follow the manufacturer’s instructions.
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?
I tried diluting in blocking solution or PBST but the results were similar! Currently running another trial using (DL-1208-.5) from Vector laboratories. Do you incubate yours 1h at RT with the secondaries or overnight at 4C with the primaries (assuming you’re colabelling with other markers)?
Usually just 1hr at room temp with secondaries.
@dmolliver have you ever seen this behavior of IB4?
Sorry, just saw this thread, hope my post doesn’t come too late. We routinely use IB4 in the secondary solution for DRG sections, usually conjugated to Alexa488. It is much “dirtier” in mouse than rat, giving a lot of speckling and more punctate staining of the cell bodies than we get in the rat.There must be more extracellular galactose glycosylation in the mouse. This noise gets better with a stronger fixation (postfix after perfusion), so I would try the strongest postfix that your antibodies can tolerate. Usually for antibodies we use little or no postfix for mouse DRG.
We have a weird problem with Thermo IB4-A647, it bleeds through into the Cy3/A555 channel, but none of our A647 secondary antibodies do this. No idea why that would be so we just use IB4-A488.
Originally we always used PBS with extra Ca2+/Mg2+ (no blocking agents) as a diluent and ran the secondaries separately in blocking buffer, but it didn’t seem to make a difference, so now we just put the IB4 in the antibody blocking solution.
Thanks @dmolliver for your insights