Methods
Tissue Samples
Protein extracts from uterus, spleen, liver, lung, cerebrum and heart control tissues were from autoptic material obtained from the Institute of Pathology from the University Hospital of Basel, Switzerland. Pancreas, kidney and colon control whole tissue homogenates were purchased from BioCat GmbH (Heidelberg, Germany). Protein extracts from breast, pancreas and kidney tumors were obtained from the Institute of Pathology of the University Hospital of Basel, Switzerland. All extracts were prepared as described before.[17]
Melanomas, kidney and lung tumors used for immunohistochemistry were part of frozen tissue microarrays (TMA) obtained from the Institute of Pathology of the University Hospital of Basel, Switzerland. References for their construction can be found elsewhere.[17] The melanoma TMA was constructed from frozen tissue samples of 34 malignant melanomas (5 primary tumors, 8 metastases from different organs and 21 lymph node metastases) and 6 control healthy cutaneous tissue samples. The kidney TMA was constructed from frozen tissue samples of 84 kidney tumors (70 clear cell renal carcinomas, 6 papillary renal carcinomas, 6 chromophobe renal cell carcinomas and 2 oncocytoma) and 10 healthy control kidney tissues. The lung TMA was constructed from frozen tissue samples of 74 lung tumors (40 squamous carcinomas, 19 adenocarcinomas and 15 undifferentiated carcinomas). Additional lung tumor samples were obtained from the University Hospital of Tours, France. The latter collection contained frozen tissues from matched samples of tumor and adjacent non-tumor tissue. They were obtained from 21 patients who had undergone lung cancer resection as their primary therapy without preoperative radiation or chemotherapy. These non-small cell lung cancers consisted of 10 adenocarcinomas, 10 squamous carcinomas and 1 undifferentiated carcinoma. The non-malignant tissue samples were taken from sites at least 3 cm away from the edge of the tumor. The histological diagnosis was determined by two pathologists.
Studies were performed in compliance with the Declaration of Helsinki and in accordance with the guidelines of the ethical committee of the University of Basel, Switzerland or with French bioethical regulations.
Western Blot Analysis
Tissue samples were thawed on ice, minced and homogenized in RIPA lysis buffer. After determination of protein concentration by a Bio-Rad Protein assay, 25 μg of protein were separated by SDS-PAGE (6%) and electroblotted to polyvinylidene difluoride membranes. Equal loading and transfer of protein was confirmed by staining the membranes with amidoblack. After a one hour blocking step in 5% milk powder in Tris-buffered saline (TBS), membranes were incubated overnight with the rabbit polyclonal antiserum pAb (3 F/4) raised against human TNW (1:750), the mouse monoclonal antibody B28–13 raised against human TNC (1:100), the V-9131 mouse monoclonal antibody against vinculin (1:2000; Sigma). After incubation for 1 hour with anti-mouse IgG or anti-rabbit IgG coupled to horseradish peroxidase, blots were developed using Super Signal (Pierce) for TNC and TNW and ECL reagent (GE Healthcare) for vinculin followed by exposure to Kodak BioMax MR Films.
Immunostaining and Quantification
Chromogenic and fluorescent detections were performed on 9 μm-thick cryosections using the Discovery XT automated stainer (Ventana Medical Systems) with standard and customized procedures, respectively. Frozen tissue slides were dried for 1 hr at room temperature, fixed for 10 min at 4°C in cold acetone and then introduced into the automate. For chromogenic stainings, slides were first blocked twice for 12 min with the AB Block reagent (Ventana). They were then incubated for 1 hr at 37°C with the mouse monoclonal 56O antibody raised against human TNW (1:1000)[18] or the B28–13 anti-TNC antibody (1:1000). They were then treated for 32 min at 37°C with a biotinylated anti-mouse secondary antibody (1:200; Jackson Immunoresearch laboratories 715–065-150) and developed with DAB Map detection kit (Ventana). Counterstainings were obtained with hematoxylin and bluing reagent (Ventana). For immunofluorescent stainings, slides were incubated for 1 hour at 37°C with anti-TNW mAb 56O (1:50), anti-TNC mAb B28–13 (1:50) or anti-CD31 (1:200; M0823, DAKO). They were then treated for 32 min at 37°C with Alexa Fluor 647 donkey anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG secondary antibodies (1:200; Invitrogen A31571 and A11029, respectively), carefully rinsed by hand and mounted with prolong Gold reagent (Invitrogen). Pictures were either acquired with a Mirax Slidescanner (Zeiss AG, Zurich, Switzerland) using a 20×/0.5 lens (0.2 μm/pixel) and converted into standard TIFF format or with a Nikon Eclipse 80i microscope equipped with a Leica DFC420 color camera. For quantification, the area of the section stained by TNW or TNC was measured using ImageJ by setting the color threshold as follows: hue 0–40, saturation 50–255, luminosity 0–200. The resulting area was divided by the total area of the section to obtain a percentage. The samples were then classified in four categories according to the amount of staining: 0%, no staining; 1%-10%, low; 11%-40%, moderate; >40%, high. In order to compare tumor type or grade with the tenascin stainings a numerical score was assigned to each category (no staining = 0, low = 1, moderate = 2, and high = 3) and the average scores for each tumor type/grade was calculated.
BMC Clin Pathol. 2012;12(14) © 2012 BioMed Central, Ltd.
