Scanscope scanner

Chick femurs from each group were dehydrated through a series of graded alcohols and embedded in low-melting point paraffin using an automated Shandon Citadel 2000. Six micrometers tissue sections were cut and stained with Weigert’s hematoxylin, followed by staining with 0.5% Alcian blue for proteoglycan-rich cartilage matrix and 1% Sirius red for collagenous matrix.
To identify human endothelial cells, tissue sections were stained for human CD31 using human specific antibody (GeneTex). In brief, after quenching endogenous peroxidase activity with 3% H₂O₂ and blocking with 1% bovine serum albumin (BSA) in 1X PBS, sections were incubated overnight at 4 °C with anti-CD31 primary antibody diluted appropriately in 1% BSA in PBS. Following primary antibody incubations, sections were incubated with biotin-conjugated secondary antibody anti-rabbit IgG (DAKO A/S; 1:100) diluted appropriately in 1% BSA in PBS. Visualization of the immune complex involved the avidin–biotin method linked to peroxidase and 3-amino-9-ethylcarbazole, resulting in a reddish brown reaction product. Sections were counterstained for light green and Alcian blue. Control staining was performed by omitting the primary antibody. Slides were digitized using high-resolution whole slides digital ScanScope scanner (Aperio Technologies, Inc.).

The excised hearts were fixed in 10% buffered formalin at 4°C for 24 hours and processed to prepare transverse, mid-ventricular, 5-μm-thick paraffin sections. These sections were stained with hematoxylin and eosin (H&E) and Masson’s Trichrome stains. The thickness of the wall of the left ventricle was measured using image analysis by scanning H&E-stained slides. High-resolution, whole-slide digital scans of all stained slides were created with a ScanScope scanner (Aperio Technologies, Vista, CA, USA). The digital slide images were viewed and analyzed using Aperio’s viewing and image analysis tools. Left ventricular thickness was measured at ×20 magnification using the linear measurement tool of Aperio’s Image Scope software (Aperio Technologies). To minimize error, thickness was measured at five randomly chosen points per heart section and averages were calculated.

Histological processing was prepared as described previously.30 The excised hearts were cleaned and fixed in 10% buffered formalin for 24 hours. The fixed samples were processed to prepare 5-μm thick paraffin sections, which were then stained with hematoxylin and eosin (H&E) and Masson’s trichrome stains. High-resolution digital scans of all stained slides were created with a ScanScope scanner (Aperio Technologies, Vista, CA, USA) and were viewed and analyzed using Aperio’s viewing and image analysis tools.