Leica bond 3 platform

Rehydrated FFPE spleen sections from Cohort 1 were stained with anti‐human CD68 (clone KP‐1, Roche, Basel, Switzerland), followed by secondary labeling with N‐Histofine (Nichirei Biosciences, Tokyo, Japan). Immunostaining procedures were performed using an automated Leica BOND‐III platform (Leica Biosystems, Wetzlar, Germany) and then counterstained with hematoxylin prior to sealing. Slides were scanned using an AxioScan Z1 (Carl Zeiss, Germany), and CD68⁺ cells were counted by a research microscopist using ImageJ (National Institute of Health, WI, USA) in a minimum area of 1 mm² of red‐pulp. To determine the number of cells per unit volume (mm³) of red‐pulp, the number of cells per unit area (mm²) was multiplied by the section thickness (5 μm = 0.005 mm).

Rehydrated FFPE spleen sections were stained manually with antibodies against Pv apical membrane antigen-1 (PvAMA1) peptide, a marker for Pv merozoites/mature stages, as described previously [29 ,50 (link)]. A dilution of 1:200 was used, and with secondary detection using N-Histofine simple stain MAX PO (Nichirei Biosciences, Tokyo, Japan). For CD68 IHC, rehydrated spleen sections were stained with a monoclonal mouse anti-human CD68 (clone KP-1) antibody (Roche, Basel, Switzerland) followed by secondary detection with N-Histofine simple stain MAX PO using an automated Leica BOND-III platform (Leica Biosystems, Wetzlar, Germany). All sections were counterstained with hematoxylin.