Scn400 slide scanner

Subcutaneous adipose tissue depots were fixed in 4% paraformaldehyde for 24 h at room temperature. Samples were then immersed in ethanol 100% before processing for paraffin embedding. To determine the adipocyte tissue diameter, paraffin sections of 5 µM were stained with hematoxylin and eosin. Images were obtained using a SCN400 slide scanner and digital image hub software 561 (Leica Biosystems, Wetzlar, Germany). Adipocyte diameter was determined using ImageJ (National institutes of health, Bethesda, MD, USA). F4/80 positive areas in the adipose tissue were randomly counted after immunostaining with F4/80 antibody (Ab6640, Abcam, Cambridge, UK). All histological observations were full blind analyzed by three individuals (S.G, M.V.H and M.R). At least 5 fields/mice were randomly selected and obtained using SCN400 slide scanner and digital image hub software (Leica Biosystems, Wetzlar, Germany).

In each patient, 2–4 myocardial biopsy samples weighing approximately 25–75 mg were gathered, of which the largest sample was analysed for histopathology, and the remaining were preserved for other use. In patients undergoing open-chest aortic valve replacement for aortic valve stenosis, or aortic valve repair for aortic regurgitation, biopsies were sampled for full width of myocardium, under direct vision by means of a Tru-Cut® biopsy needle (CareFusion, Waukegan, IL) in the anterior wall. Patients undergoing mitral valve repair for mitral valve regurgitation by either Port-Access or Da-Vinci minimal-access had samples taken under endoscopic vision taken by a surgical scissor from the anterior septum. Samples were immediately fixed in 10 % buffered formalin, embedded in paraffin, sectioned, and stained with picrosirius red. Stained sections were digitalized with a SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany). Quantification was performed using TissueIA software (Leica Biosystems, Dublin, Ireland). After elimination of artifacts and perivascular fibrosis, area occupied by interstitial fibrosis was expressed a percentage of total endomyocardial area. Four different histological slices were analyzed per patient and the average of the quantification of the different specimens was considered the final value of fibrosis for the patient.

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.