E-cadherin and β-catenin mark EBs that arise in the rat anemic spleen. (A) Rat spleen weight defined after isolation from control (PBS; n = 4) and anemic animals (2, 5, and 16 days after final injection with PHZ; n = 4) reveals spleen weight increase by fourfold from ∼0.5 g to 2 g within 48 hours after PHZ treatment. (B) Representative flow cytometry plots of isolated splenocytes stained for CD71 and HIS49, revealing that splenocytes from control (PBS) rats barely contain CD71⁺HIS49⁺ EBs (∼3%), which increase up to 49% in response to anemia (PHZ 48 hours). (C) Representative IHC staining for E-cadherin (brown) on rat spleen sections obtained from control (PBS) and anemic (PHZ 48 hours) rats revealing E-cadherin to mark macrophage-typed cells in the red pulp in control conditions and to mark EB clusters that develop in the red pulp in response to anemia. (D) Representative IHC staining for β-catenin (brown) in the rat spleen, 48 hours after PBS (control) or PHZ treatment, revealing β-catenin protein expression to mostly mark endothelial cells in control conditions while marking EBs that arise in the red pulp in response to anemia.