Tissuefaxs microscope

Three days after catheterization and imaging procedures, each rat was euthanized. Livers were harvested and tumor-containing segments resected for microtome sectioning. 5 μm slices through the center of each tumor were used for both hematoxylin and eosin (H&E) and Prussian blue staining (providing confirmation of ferrofluid-containing microsphere delivery). Additional slices were used for rat anti-CD34 staining and microvessel density (MVD) measurements. All slides were digitized at x200 optical magnification using a TissueFAXS microscope (TissueGnostics GmbH, Vienna, Austria). Post-processing was performed using the HistoQuest software package (TissueGnostics GmbH). Post-processing steps involved digitally stitching together the individual images acquired with the TissueFAXS microscope to produce a signal image depicting the entire specimen on each slide. From this compiled image, the tumor was selected with manual delineation of a region of interest (ROI). Tissues within this ROI were evaluated with an automated threshold analysis approach to identify areas stained brown thus indicating positive staining of CD34. We then calculated the percentage of the ROI stained positively for CD34, our quantitative estimate for MVD. The HistoQuest software was used to measure the tumor area within each immunohistochemistry slide.

The harvested tumor tissues were fixed with 10% neutral formalin 4 solution and then submitted to Mouse Histology and Phenotyping Laboratory (MHPL, Northwestern University, USA) core for Gram-positive, H&E and TUNEL staining with 5 μm slice thickness. All slides were analyzed using a TissueFAXS microscope (TissueGnostics GmbH, Vienna, Austria). The quantitative analysis of TUNEL-positive cells in the tissues was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).

After post-procedural imaging, rabbits were recovered and survived for 24 hours before they were sacrificed. Afterwards, sections of the liver, stomach and lung were harvested and preserved in formalin for subsequent paraffin embedding, Prussian blue staining (demarcating iron-oxide accumulation) and H&E histological staining (1 cross section taken through the center of each tumor) as well as anti-CD31 immunohistochemistry (IHC) (3 separate cross sections through each tumor, each section within roughly 4 mm of the geometric centroid of the lesion) for microvessel density measurements. The subsequent slides were then imaged using a TissueFAXS microscope (TissueGnostics GmbH, Vienna, Austria). Anti-CD31 IHC slides were analyzed using associated HistoQuest software (TissueGnostics GmbH) to determine percentage of areas positive for CD31 staining within the tumor tissues (serving as a quantitative measure of microvessel density).