Expression Pattern of Aquaporin 1 and Aquaporin 3 in Melanocytic and Nonmelanocytic Skin Tumors

Giovana Osorio, MD, PhD; Teresa Zulueta-Dorado, MD, PhD; Patricia González-Rodríguez, PhD; José Bernabéu-Wittel, MD, PhD; Julian Conejo-Mir, MD, PhD; Reposo Ramírez-Lorca, PhD; Miriam Echevarría, PhD

Disclosures

Am J Clin Pathol. 2019;152(4):446-457. 

In This Article

Materials and Methods

Biopsy Specimens From Skin Tumors

In this study, we performed immunohistochemical analysis of AQP1 and AQP3 expression on tissue sections of biopsy specimens from 70 patients with various skin tumors, as shown in Table 1. Samples of all tumors were collected by surgery and kept as paraffin-embedded samples within the Department of Pathology, Hospital Universitario Virgen del Rocío (HUVR), Seville, Spain. Normal skin samples from healthy borders around the lesions were also analyzed. The tumors were staged according to the TMN classification system and histologically classified according to the World Health Organization guidelines.[11] Tissue slides were H&E stained and evaluated for diagnosis by two independent pathologists.

Written informed consent was obtained from all participants. The study followed the tenets of the Declaration of Helsinki for research on human subjects and was approved by the ethics committee of HUVR.

Immunohistochemistry

To set the immunohistochemistry conditions to detect AQP1 and AQP3, different dilutions of the antibody and developing times were tested on skin samples. All samples examined were obtained from formalin-fixed, paraffin-embedded pieces.

Tissue slices of 2 to 5 μm were cut with a microtome and mounted on microscope slides. The immunohistochemical procedure started by removing the paraffin from the samples by immersion in xylene and rehydration through a series of decreasing dilutions of ethanol. Blocking of endogenous peroxidase activity was done by preincubation of samples in 3% H2O2. Heat-induced epitope retrieval was carried out by incubation of tissue sections at 65°C for 1 hour in sodium citrate (10 mmol/L, pH 6), and then blocking of unspecific binding was done by incubation for 1 hour in a phosphate-buffered saline solution with 10% bovine serum and Triton X-100 at 0.3%. Rabbit polyclonal anti-AQP1 (cat. ab15080, RRID: AB_2056839, 1:500 dilution; Abcam) or anti-AQP3 (cat. sc-20811, RRID: AB_2059551, 1:50 dilution; Santa Cruz Biotechnology) was used. The next two steps were EnVision + Dual Link System-HRP (DakoCytomation) that contains goat anti-rabbit immunoglobulins conjugated to peroxidase-labeled polymer and 3,3′-diaminobenzidine (DAB) substrate/DAB chromogen for the development of brown precipitates. Qualitative analysis of signal level was performed according to the following criteria: absence of brown precipitate indicated a "negative" result for immunoreactivity, and the presence of brown precipitate over specific cell areas was considered a "positive" result. Counterstaining with hematoxylin was done, and sections were analyzed side by side with the pathologist (T.Z.-D.) by two independent observers (G.O. and M.E.). Sections were photographed using an AX70-Olympus microscope equipped with an Olympus DP10 camera. Omitting the primary antibody produced no staining.

Statistical Analysis

Data analysis was performed using the Statistical Package for Social Sciences (SPSS), version 16.0. Continuous variables were described with the mean and standard deviation. Categorical data were summarized with the absolute and relative frequencies of each category. The expression of AQPs for each neoplasm and anatomical location was analyzed by χ 2 or Fisher exact test as appropriate. The α error was set at .05.

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