Figure 1.

Ab-oligo cyCIF strategies. Multiple methods of fluorescent labeling of Ab-oligo conjugates have been investigated including (A) a complementary imaging strand (IS, 26 nt) with a single fluorophore and photocleavable linker (PCL) and (B) a complementary IS (26 nt) with two fluorophores and two PCLs that hybridize to the docking strand (DS, 28 nt) on the antibody. (C) An amplification strategy has been developed and optimized that utilizes an amplification strand (AmpS, 198 nt) that hybridizes with DS (28 nt) and has an unhybridized region that can hybridize up to ten amplification IS (Amp IS, 15 nt) that each contain a fluorophore and PCL for increased signal intensity. (D) The oligonucleotide conjugation strategy was also extended to secondary antibodies to permit Ab-oligo cyCIF of sensitive primary antibodies where DS (28 nt) are conjugated to secondary antibodies and detected using complementary 2× PCL-FL IS (26 nt).