Tissue ia

Melanoma xenografts were fixed in 4% formaldehyde for 24 hours and embedded in paraffin. Following deparaffinization, inactivation of endogenous peroxidases, antigen retrieval in citrate buffer and aspecific binding blocking, 5 μm sections were incubated overnight at 4°C with the primary antibodies for phospho-ERK (Cell Signaling Technology, ref. #4370, 1:200 dilution), Ki-67 (Cell Signaling Technology, ref. #9027, 1:600 dilution) or Cleaved Caspase 3 (Cell Signaling Technology, ref. #9661, 1:300 dilution). Consequently, sections were incubated at room temperature for 30 minutes with Envision anti-rabbit secondary antibody (Dako, ref. #K4003) and stained with diaminobenzidine for 5 min (Dako, ref. #K3468). Stained slides were then digitalized using a SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany) at X20 magnification and analyzed using TissueIA (Leica Biosystems, Dublin, Ireland). The quantification algorithm was run in the viable part of the tissue samples to detect stained area and tissue area. A staining index was calculated as the average staining intensity of the positive pixels multiplied by the positively stained area fraction. Regions of interest corresponding to necrotic areas were manually defined, and for each slide necrotic fraction was calculated as the sum of all necrotic areas divided by the total tissue area.

Digitalization of the scanned slides was done at a 20x magnification by SCN400 slide scanner (Leica, Wetzlar, Germany). Scanned slides were analyzed using Tissue IA (Leica Biosystems, Dublin, Ireland). The tumour tissue was delineated manually and folds, bubbles and dirties were excluded from the analysis. Colour deconvolution was applied using hematoxylin and DAB matrices of the software. Nuclear algorithms were applied for cyclin D1 and Ki67 immunostaining, keeping the parameters constant for all slides. Thresholds were adjusted for tissue and DAB detection and also for nuclear segmentation. Percentage of nuclei with positive staining and corresponding staining intensity were generated. Cyclin D1 immunoreactivity was evaluated as: negative; grade 1, focal staining in less than 25% of tumour cells; grade 2, staining in 25–50% of tumour cells; and grade 3, diffuse staining in more than 50% of tumour cells [18 (link)]. Ki67 staining was scored according to percentage of cells showing nuclear staining, as less than 1%, 1 to 5%, and 5 to 20% [18 (link)].
For CK19 and HBME-1, a distinct staining in cytoplasm and cell membranes was considered as significant. Galectin-3 showed nuclear and cytoplasmic staining in thyroid carcinomas. The staining in the normal thyroid tissue surrounding the follicular adenomatoid nodules was also evaluated at the same time as the tumour.

Two paraffin sections of jejunum of 5 µm per mice were stained with primary antibody against perilipin-3 at dilution 1:500 (catalog abs482, Millipore, Darmstadt, Germany) followed by secondary antibody donkey anti-rabbit Dylight 594 at dilution 1:1000 (SA5-10040, Thermofisher, Waltham, MA, USA). Nuclear counterstaining was performed with Hoechst 33342. Images were captured by a Pannoramic 250 Flash III (3Dhistech, Budapest, Hungary). Percentage of staining was calculated with Visiopharm software version 6.6.3.
For hepatic lipid staining, frozen liver sections were sliced at 5 µm, treated with oil red O and scanned as previously described [27 (link)]. The lipid area was determined on whole sections using the imaging software TissueIA (version 2.0.3, Leica Biosystems, Dublin, Ireland). Pixels corresponding to the oil red O staining were selected to create a color profile. Total tissue area was defined by setting the tissue intensity threshold at 210 (grey value). Results were expressed as stained area (below threshold)/tissue area (below threshold). Two representative tissue pieces were analyzed for each mouse.

Sections were digitalized at a 20x magnification by SCN400 slide scanner (Leica, Wetzlar, Germany). The tumor tissue in each section was delineated manually and care was taken to exclude tissue folds, bubbles and artefacts from the analysis. Scanned slides were then quantified using Tissue IA (Leica Biosystems, Dublin, Ireland).
Quantification included application of algorithms for nuclear (cyclin D1 and pHH3) or cytoplasmic (bcl-2 and cleaved caspase-3) immunostaining. Color deconvolution was applied to each pixel using hematoxylin and DAB matrices of the software. On the hematoxylin matrice, nuclear parameters (size, heterogeneity, strength of nuclear counterstaining, nuclei density) and cellular parameters (size and cell radius) were adjusted to determine the adequate segmentation. On the DAB matrice, a threshold was adjusted for DAB detection according to intensity (grey values from 0 to 255). These parameters were kept constant throughout the study for each immunostaining.