Seeking to stain T cells for surface-markers and ICC for cytokines/Foxp3, do I need to use FC-bock before surface-staining and what is recommended? (eg. Heat-inactivated human serum). I do not include ısotype control but instead use FMO's.
Fc block might not be necessary. Blocking for 30 min on ice with 10% human serum and 1% BSA in PBS is usually more than sufficient. If you are worried though, Fc block from BD works really well. You only incubate for just a few minutes, and then leave the Fc block in while staining. I think it is always in good practice (and honestly an absolute necessity) to do isotype controls, particularly for the intracellular staining. Permeabilizing cells usually leads to increased autofluorescence and it can be harder to distinguish between positive and negative cells. Hope this helps!
Human serum can work, or antibodies for Fc receptors.
I would suggest, since you have multiple staining steps, checking Isotype controls of ICC antibodies at list once just to see you don’t get nonspecific binding.
I usually block with Cohn II fraction (sigma) at 1mg/ml for 10min. As you may know monocytes are good to study blocking in PBMC for extracellular markers so check on that.
In intracelllar stainings, you also may want to check on a isotype control as suggested at least once to get a feel. The best control, however, are non-conjugated antibodies of the same specificity and subsequent addition of conjugated antibody should yield no fluorescence. However, this is usually more pricy.
Fc block might not be necessary. Blocking for 30 min on ice with 10% human serum and 1% BSA in PBS is usually more than sufficient. If you are worried though, Fc block from BD works really well. You only incubate for just a few minutes, and then leave the Fc block in while staining. I think it is always in good practice (and honestly an absolute necessity) to do isotype controls, particularly for the intracellular staining. Permeabilizing cells usually leads to increased autofluorescence and it can be harder to distinguish between positive and negative cells. Hope this helps!
Which FC-block do you use for staining bovine monocytes? Why AB human serum?
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