Gam hrp

Fixed lungs, livers, and tumors were processed using a Diapath automatic processor. In brief, tissues were dehydrated through exposure to 70% ethanol for 60 min, two 90-min washes in 95% ethanol, and three 60-min washes in 99% ethanol. Tissues were then cleared through three 90-min washes in xylene. Finally, the tissues were subjected to three 1-hour immersions in paraffin. Paraffin-embedded samples were stored at room temperature until cutting. Hematoxylin and eosin staining (Diapath) was performed on serial sections to assess histological features. For immunohistochemistry, paraffin was removed with xylene and sections were rehydrated in graded alcohol. Antigen retrieval was carried out using preheated target retrieval solution for 30 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in distilled water for 10 min at room temperature. The tissue sections were blocked with FBS in 1× PBS for 60 min and then incubated overnight with primary antibody, anti-human nuclei diluted 1:200 (Millipore, MAB4383 clone 3E1.3). The primary antibody was detected using a polymer detection kit (Microtech, GAM-HRP) followed by a diaminobenzidine chromogen reaction (Vector Laboratories, Peroxidase substrate kit, DAB, SK-4100). All sections were stained with Mayer’s hematoxylin and visualized using a bright-field microscope.