This document summarizes the immunohistochemistry protocol used to analyze paraffin embedded human lung cancer tissue samples for carbonic anhydrase IX (CA IX) expression. The protocol involved antigen retrieval using citric acid, blocking of endogenous peroxidase, blocking with BSA, overnight incubation with a CA IX monoclonal primary antibody at a 1:200 dilution, incubation with an HRP-labeled secondary antibody, DAB staining, hematoxylin counterstaining, dehydration, clearing and mounting before visualization under a microscope.