Hrp conjugated polymer anti rabbit

Cervical (CR802) and esophageal (ES482) tissue arrays including cancer and normal tissues as well as a universal normal tissue array (UNC241) were obtained from US Biomax, Inc. (Rockville, MD). Tissue arrays were deparaffinized with xylene and hydrated with graded ethanol solutions. Antigen retrieval was performed using citrate buffer, reduced pH, and endogenous peroxidases were quenched by incubating slides in 3% H₂O₂. The arrays were blocked with 2.5% bovine serum albumin (Sigma) in PBS, and incubated overnight at 4°C with 2 μg/ml rabbit anti human TMPRSS11d (HAT Ab1) (ab127031, Abcam, Cambridge, MA) or rabbit anti human TMPRSS11d (HAT Ab2) (HPA052834, Sigma) in a humidity chamber. All washing steps were performed using PBS. As a negative control, non‐immune rabbit IgG (2 μg/ml) (NeoMarkers, Fremont, CA) was used. Bound antibodies were visualized using biotin‐conjugated anti‐rabbit (Vector Laboratories, Burlingame, CA) secondary antibodies, and a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) or HRP conjugated Polymer Anti‐Rabbit (Dako, Carpinteria, CA). 3,3′‐diaminobenzidine (DAB) was used as the substrate (Sigma) and arrays were counterstained with hematoxylin. All microscopic images were acquired on a Zeiss Scope A.1 using digital imaging.