Which fluorochrome-conjugated secondary antibodies do you recommend for IF and what are the differences?

At CST, we recommend fluorochrome-conjugated F(ab')2 fragment secondaries as they reduce the non-specific background that is usually associated with their whole molecule counterparts. Whole molecule secondaries have the highest capacity for dye conjugation and, thus, are likely to yield the brightest signal. Their major downside is background, as many cell types express Fc receptors that these are likely to stick to without sufficient blocking.
F(ab')2 have had their Fc regions removed through pepsin digestion, leaving two antigen binding domains.
F(ab')2 antibodies have reduced capacity for dye conjugation which may result in a slightly weaker signal when compared to their whole molecule counterpart. Despite this, we find this format to provide an optimal balance between signal and noise.
Monovalent F(ab) fragments are highly penetrant due to their small size, but also yield extremely low intensity. In our experience, these are best utilized as blocking reagents in their unconjugated form and we do not recommend using them for secondary detection.

Last updated: February 27, 2024