K4061

Hemi-mandible specimens were decalcified in 10% EDTA at 37 °C for 7 days before routine processing. Fixed tissues and organs were embedded in paraffin wax, sectioned in 3 µm-thick slices and processed for hematoxylin and eosin and Masson trichrome stainings. Anti-human-vimentin immunohistochemistry (IHC) was used as a specific, accurate and sensitive method to track the transplanted human ASC [74 (link)], and anti-human Ki-67 IHC to estimate their proliferation. Staining of 3-µm sections of paraffin-embedded specimens was performed after antigen retrieval for 30 min (Ptlink, low pH, ref K8005, Dako, Les Ulis, France), using anti-vimentin antibody (M0725, mouse monoclonal antibody, Dako, Les Ulis, France, dilution 1/50, 50 min at RT) and the ARK (Animal Research Kit, Dako, Les Ulis, France) or anti-Ki-67 antibody (ref K4061, mouse monoclonal antibody, Dako, Les Ulis, France, dilution 1/50, 50 min at RT). Staining was carried out with Dakostainer automated system using the envision secondary step and DAB as chromogen. Proliferation index (number of Ki-67 positive ASC on total cells) and cell density (number of vimentin positive ASC on total cells) were assessed on scanned slides (Scanner Pannoramics 250, 3DHistec, Brignais, France) and analyzed by manual counting on the region of interest with a panoramic viewer.

Sections (4 µm thick) were prepared from parafin-embedded intestinal tissues. After deparaffinization, antigens were recovered by incubating the slides in citrate buffer (pH 6.0) for 20min at 95°C. Endogenous peroxidase was blocked with 3% H₂O₂ for 10 min to reduce nonspecific binding. Sections were then incubated with TNF-α (ab270264, Abcan) or iNOS (ab3523, Abcan) for 2h. Sections were then incubated for 30 min with polymer (K4061, DAKO). Antibody binding sites were visualized by incubating the samples with diaminobenzidine–H2O2 (DAB, DAKO) solution. Sections incubated with antibody diluent, without a primary antibody were used as negative controls. The amounts of DAB products from immunostaining were estimated from digital images of at least ten different areas of each section (from 4 specimens per group) at 400x magnification using Adobe Photoshop sofware. The results are expressed as the percentage of immunopositive area, calculated by dividing the DAB-positive staining (immunostaining-positive pixels) by the number of pixels per total tissue image multiplied by 100, as previously described (16 (link)).

During the 2-year study period (2012-13), all 78 cases reported as HP, SSA/P or TSA were extracted from the pathology archives of hospitals affiliated with Shiraz University of Medical Sciences. All slides were reviewed by a GI pathologist (BG) and re-classified according to the last version of the WHO (2010, 4th edition) (5 ).
Demographic characteristics of the patients and size of the polyps were recorded from either the pathology or endoscopy reports.
Paraffin blocks of all cases were extracted from the pathology archives and prepared for immunohistochemistry for MLH-1. Routine immunohistochemistry staining was performed for MLH1 using the primary prediluted antibody (mouse monoclonal, Biocare). For this purpose, after deparaffininzation with xylol and blocking endogenous peroxidase with H2O2 and antigen retrieval with Tris EDTA, sections were incubated with primary Ab and then envision (k4061,Dako) and diaminobenzidine (DAB), they were counterstained with Hematoxylin. Negative nuclear staining was interpreted as MLH1 gene deletion.