Ta 125 hdx

Following euthanasia, the sternum and spleen were collected and fixed in 10% neutral buffered formalin, followed by decalcification of the sternum in 10% formic acid. The tissues were embedded in paraffin and 4-μm sections were stained with hematoxylin and eosin (H&E) or were used for IHC analysis. IHC was performed on formalin-fixed, paraffin-embedded (FFPE) tissues sectioned at 4 μm. All assay steps for GATA1, CD3, and MPO, including deparaffinization, rehydration, and epitope retrieval, were performed on the Ventana Discovery Ultra autostainer with Ventana Reaction Buffer (Ventana, 950-300) rinses between steps. All assay steps for B220/CD45R and Pax5 were performed on the Bond Max with Bond wash buffer (Leica, AR9590) rinses between steps. Antigen retrieval and incubation with the primary antibody were performed. Labeling was visualized with streptavidin conjugated to horseradish peroxidase (Thermo Fisher Scientific, TS-125-HR; 10 minutes) and substrate containing the chromagen DAB (Thermo Fisher Scientific, TA-125-HDX; 5 minutes).

Brains were taken and stored in 10% neutral formalin. After fixation, tissues were processed in paraffin blocks and sectioned at 4 μm, and immunohistochemistry was performed on paraffin-embedded sections with a monoclonal mouse anti-nucleoprotein influenza A virus antibody (Argene; 11-030; pronase 0.05% retrieval solution, 10 min at 37°C, antibody dilution of 1/50, incubation overnight at 4°C). The immunohistochemical staining was revealed with a biotinylated polyclonal goat anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP; Dako; LSAB2 system-HRP, K0675) and the diaminobenzidine HRP chromogen (Thermo Scientific; TA-125-HDX). Negative controls comprised sections incubated either without specific primary antibody or with another monoclonal antibody of the same isotype (IgG2).