Immunohistochemical Staining of Feline Tissues

To test whether the antibody that positively labeled feline normal and neoplastic epithelial cells on flow cytometric analysis (the goat polyclonal R&D EpCAM) could detect a protein with expected membrane-associated expression in normal and neoplastic feline tissue, we performed IHC staining on formalin-fixed paraffin-embedded tissue. This staining was initially performed on an array prepared from tissues from a healthy cat by the Histology Laboratory in the Animal Health Diagnostic Center at Cornell University. The array contained the following tissues: liver, renal cortex, oral mucocutaneous junction, pancreas, stomach (fundus), duodenum (distal to papilla), jejunum, colon, lung, adrenal gland, thyroid gland, pituitary gland (pars nervosa), brain (frontal lobe), testes, outer aorta, heart, mandibular and mesenteric lymph node, tonsil, spleen, thymus, and skeletal and smooth muscle. Additional sections of normal feline colon were used to optimize the immunostaining procedure. Immunostaining for EpCAM was then performed on two feline mammary carcinomas, one of which had adjacent, non-neoplastic mammary epithelium as an internal control and normal cutaneous epithelium, and an oropharyngeal squamous cell carcinoma with normal oral tissue obtained from a healthy cat as a negative control. The mammary carcinomas were typed as a grade II tubulopapillary carcinoma and a grade III tubular carcinoma (31). The mammary carcinomas were archived samples in the Histology Laboratory, whereas the oropharyngeal squamous cell carcinoma was obtained from prospectively collected tissue that was placed in the Cornell University Biobank. The latter protocol was approved by the Institutional Animal Use and Care Committee at Cornell University (#2005-0151). For both initial and optimized procedures, 4 μm–thick sections of formalin-fixed, paraffin-embedded sections were deparaffinized in xylene and rehydrated with graded ethanol, followed by antigen retrieval steaming for 20 min in either Tris-EDTA (pH 9.0) (initial protocol) or sodium citrate (10 mM, pH 6.0) (optimized protocol). Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in distilled water for 10 min. In the optimized procedure, non-specific staining was blocked with a mixture of 10% rabbit serum and 2 × casein for 1 h at room temperature. Immunostaining was then performed using the ImmPRESS HRP Anti-Rabbit Ig (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA) according to kit instructions. The latter kit used 3,3′ diaminobenzidine as a chromogen. In the initial procedure, sections were incubated with various dilutions of the goat polyclonal R&D EpCAM antibody overnight at 4°C (1:10–1:200). In the optimized procedure, the antibody was incubated at a 1:10 dilution for 3 h at room temperature, followed by 20 h at 4°C. Negative controls were run in parallel by replacing the primary antibody with goat immunoglobulin G (IgG) at an equivalent final concentration. After washing three times in PBS with 0.05% Tween 20, the sections were incubated with biotinylated rabbit anti-goat IgG (1:200, Vector Laboratories, Burlingham, CA) for 1 h at room temp, followed by streptavidin-horseradish peroxidase conjugates (Ready to use, Vector Laboratories) for 20 min at room temperature. For some tumors with the optimized procedure, Nova Red (Vector Laboratories,) was used as an alternative chromogen to visualize antigen localization and sections were lightly counterstained with hematoxylin. Stained sections were examined with an Olympus AX 70 compound microscope, equipped with MicroFire camera and PictureFrame for image processing and capture (Optronics, Goleta, CA) or an upright microscope (BX40, Olympus Corporation, Life Science Solutions, Center Valley, PA), equipped with a digital camera (Olympus, model UC90), using cellSens software (standard 1.18, Olympus).