I tried earlier with 5% Natural Goat Serum but I see a lot of background fluorescence in cells stained with secondary antibody only. I spoke with a few company represenatatives apparently they dont have any experience with IF. They offere solutions for Flow cytometry. I have seen people recommending AB serum. Any answers would be appreciated. Thanks.
Hi Junaid Ur Rehman
You can use a combination of both NGS and BSA (5%), as it would help in providing more effective blocking when compared to using them alone.
BSA and NGS can bind to the Fc region of IgG, as they have different properties that can complement each other. BSA is a small protein that is relatively easy to penetrate into cells, while NGS is a larger protein that can more effectively block Fc gamma receptors on the cell surface.
Thanks,
Hi Junaid
Yes, this is a problem for those of us who work with fluorescence that can be solved with the use of flow cytometry, if you don't need the beauty of an image, you know.
We use fetal bovine serum and for the negative controls we recalibrate the software very well to avoid that background noise. Have you thought about the possibility of not using the serum, that is, is it necessary to use the serum for that photo?
Greetings.
Hi Xavier,
What percentage of FBSdo you use. I use NGS cos my secondary antibodies are raised in goat. Setting exposure time where you see no fluorescence with control is a good idea.
Hi Junaid Ur Rehman
I suppose You should try solution from cytometry, like FC block. Since, the principles of blocking the same it should work
Although the world of colors of fluorescence microscopy is just as beautiful, it has different secrets than the explosion of colors of flow cytometry, you just have to discover them..... :-)
Dear Xavier Ferez Ruiz
What do You mean? What kind of secrets?
FC Block for flow cytometry dosen't have fluorophore. It is used only for block but not for stain. Technically it's just antibody to FcγII and FcγIII
Hello Andrey.
The answer was for colleague Junaid...
I have used quite a bit of literature in my reply to my colleague Junaid... Sometimes it doesn't hurt to use some literary figures in Pure Science.
@For those of us who have used both fields, flow cytometry and fluorescence microscopy, we have always encountered the problem of "background noise", that is, "false positives" even when we do not use fluorochromes or an FC Block that could have a natural fluorescence. But this above all in fluorescence microscopy, as was the case about which our colleague Junaid asked us.
I'm sure that close to him he will have some more experienced colleagues who can help him, so cheer up Junaid.
Well, to give a more specific and technical answer, you would have to know what cells you are using, what fluorochromes you are using, what microscope you are using, how your lasers are configured, what image analysis program you are using to filter out that background noise. And finally, fine-tune the protocol in your own laboratory because, as you already know, sometimes the protocols do not turn out the same and need adjustments.
Forgive my English and best regards.
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