Strengths of our study were as follows: first, we used robust staining controls; second, we determined HIF-1α immunopositivity as a continuous variable allowing analyses that included adjustments for cell density; third, we developed and validated the use of the paired same area serial analysis; and fourth, we assessed HIF-1α functionality in the CRC tissues by demonstrating coincident expression with CA-IX, an established HIF-1 transcriptional target. The gene discussed is CA9; the disease is colorectal carcinoma.