Antibody specificity was confirmed on formalin-fixed cell pellets of MCF7 cells (Supplementary Fig. 1, see section on supplementary materials given at the end of this article). Patient tissue samples were run on Leica’s Polymer Refine Detection System (DS9800) using their standard template on the automated Bond-III platform. MCF7 cells were cultured in estrogen-rich complete DMEM media with 10% BS and glutamine. Post-siRNA knockdown of ZMIZ1 were used as negative control to ensure antibody specific. Dewaxing and re-hydration prior to IHC were automated on the Leica ST5020, along with the post-IHC dehydration and clearing. Sections were mounted using Leica’s coverslipper CV5030. The specific antibody targeting ZMIZ1 was purchased from R&D Systems (AF8107) and used at a concentration of 2 µg/mL (1:250 dilution). The sodium citrate pre-treatment was run at 100˚C. The secondary (post-primary) was rabbit anti-sheep from Jackson ImmunoResearch (r313-005-003), diluted 1:500. DAB Enhancer was included as an ancillary reagent (Leica, AR9432).
Patient tissue samples were processed as described for the MCF7 fixed cell pellets. ER-α antibody was purchased from Novacastra (NCL-ER-6F11/2) and samples processed as previously described (Bruna et al. 2016 (link)).
Patient tissue samples were processed as described for the MCF7 fixed cell pellets. ER-α antibody was purchased from Novacastra (NCL-ER-6F11/2) and samples processed as previously described (Bruna et al. 2016 (link)).