Immunohistochemical Analysis of C/EBP Isoforms

First, 4 µm thick TMA cores were deparaffinized and endogenous peroxidase activity was blocked using 0.3% H₂O₂ in methanol for 15 minutes. Next, slides were blocked using Ultra V Block (#TA-125-UB, Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at room temperature. Slides were then incubated with primary antibodies against C/EBPβ or C/EBPγ in PBS at 4 °C overnight (C/EBPβ 1:500 #GTX100675, GeneTex, Irvine, CA, USA; C/EBPγ 1:500 #abx103894, Abbexa Ltd., Cambridge, UK). The C/EBPβ antibody binds to the transcriptionally active C/EBPβ isoforms LAP and LAP* but not to the inactive isoform LIP. Only one isoform C/EBPδ and C/EBPγ is known to date. The next day, slides were incubated with secondary HRP-linked goat-anti-rabbit antibody (#DPVO55HRP, ImmunoLogic, Duiven, NL, USA) for 30 minutes at room temperature and stained using 3,3′Diaminobenzidine (Bright DAB #BS04-999, ImmunoLogic) with hematoxylin (1:10 in demineralized H₂O) counterstain. To assess C/EBPδ expression, the TMA and scoring published previously were employed [15 (link)].