Immunostaining of Lung and Liver Tissues

Four-micrometer sections of formalin-fixed, paraffin-embedded lung and liver tissues were immunostained for cytokeratin 19 (A53-B/A2, mouse monoclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA) using the standard avidin-biotin complex immunoperoxidase method (Vectastain ABC-Elite; Vector Laboratories, Burlingame, CA) as described previously^{40 (link)}. Briefly, the sections were incubated with a primary antibody at a dilution of 1:50. Subsequently, the sections were incubated with biotinylated anti-mouse antibody (Vector Laboratories, Inc., Burlingame, CA) diluted 1:250 and further incubated with VECTASTAIN Elite ABC reagent. The sections were subsequently processed with peroxidase substrate solution. The sections were then counterstained with hematoxylin. The sections were examined under light microscopy. The cells were considered to be positive for cytokeratin 19 expression in the cytoplasm if tumor cell cytoplasm stained a brown color above the cytoplasmic background.