Quantitative Immunohistochemistry of MZF1 and Lin28A

Tissue microarrays (TMAs) were purchased from Pantomics (BRC961, BRC962 BRC1501, BRC1502, BRC1503), Richmond, CA, USA). Additional tissue samples of normal breast tissue were produced and processed as previously published^{57 (link)}. FFPE cell-blocks were prepared as in Cabezon et al.^{58 (link)}. Five micrometer sections were stained with primary antibody against MZF1 (Abcam ab64866, 1:1000, Cambridge, UK) or Lin28A (Abcam ab46020, 1:1000). The sections were scanned and assessed in an ACIS^® III Instrument (DAKO). IHC quantification was done by ACIS III-assisted analysis. The stained sections were scanned and brown intensity was quantified with manually placed 40× regions (3 per section/core) and a color threshold customized for MZF1.