can you provide me with the best p16/ki67 dual staining protocol for FFPE tissue (cervical)? and what are the best antibody brand for that?
I have not performed this but similar combinations before. I guess you are working on human material, and that you are familiar with IHC/IF and heat induced epitope retrieval (HIER). To make it easy, I would go for IF and use monoclonals from different species and recommended by NordiQC for use on patient material.
Titer both antibodies first for optimal dilution in your setting (depending on HIER buffer and amplification technique). I use 0.05% citraconic anhydride pH7.4, Aldrich #125318, which commonly is inferior to citrate based buffers; Namimatsu et. al., 2005) prior blocking with 5% serum matching the source of your secondary, combined primaries overnight at 4 °C, wash, combined fluorophore conjugated secondaries (e.g. Invitrogen or JIR) including DAPI for 2h at Rt, wash, mount with PVA containing DABCO (Aldrich #10981), let is set for at least 1h before watching.
For Ki-67 I use Ms mAb clone MIB-1 (Dako) and Rb mAb clone SP6 (Invitrogen), at approx. 1/200-400. See also https://www.nordiqc.org/downloads/assessments/84_1.pdf
For p21 I would follow NordiQC and go for clones JC2, MX007, 6H12 or E6H4 (I would choose MC007). See
https://www.nordiqc.org/downloads/assessments/134_59.pdf
Good luck!
Olav
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