Nanozoomer 2

Immunohistochemical staining was quantitatively evaluated using computer-assisted image analysis software (Visiopharm, Hoersholm, Denmark) as described previously (14 (link)). In brief, the slides were scanned using a whole-slide scanner (NanoZoomer 2.0, Hamamatsu Photonics, Hamamatsu City, Japan) and imported into Visiopharm software (for Windows 7, version 4.5.1.324) using the TMA workflow. Staining intensity was categorized as 0, 1+, 2+, or 3+ according to the distribution pattern across cores. A brown staining intensity (0-negative, 1-weak, 2-moderate, or 3-strong) was obtained using the predefined algorithm and optimized settings. The overall immunohistochemical scores (histoscore) for AGR2, BRD7, and POM121 were expressed as the percentage of positive cells multiplied by their staining intensity (possible range, 0–300). For the survival analysis and hierarchical clustering, the expression values were dichotomized (High vs. Low, described in superscript) using cut-off values affording the most discriminative power (histoscore of 39 for AGR2, 46 for BRD7, and 204 for POM121). PAUF protein expression was evaluated as previously described (4 (link)) to compare with that of the three proteins identified.

Cardiac and aortic tissues were fixed with 4% (w/v) paraformaldehyde overnight. Then the tissues were embedded in paraffin, cut into 5 μM slices, and stained with hematoxylin and eosin or oil red O. Images were captured using NanoZoomer 2.0 (Hamamatsu, Japan).

For bright-field and fluorescence, slides were scanned with Nanozoomer 2.0 RS Hamamatsu (with the fluorescence imaging module for tumor slides stained with anti-CD34, anti-αSMA, anti-cleaved-caspase-3, or anti-Ki67). Absolute numbers of CD34-positive vessels present within 1.5 mm² of the tumor area and percent of αSMA/CD34-positive staining per 0.76 mm² area and number of HIF-1α-positive cells per 0.8 mm² area for each tumor slide were quantified by optical counting. Automatic cell counts of Ki67-positive and cleaved caspase-3-positive cells and automatic GLUT-1 and pimonidazole intensity were determined per 1.5 mm² of the tumor area with Image J Software (NIH, Bethesda, MD).

Adult (10 week-old) controls and Vangl2 cKOs mice were perfused transcardially with PB followed by 4% PFA in PBS; the brains were removed and postfixed in 4% PFA for 48 hr at 4°C before sectioning. All coronal sections were stained with cresyl violet, scanned with Hamamatsu NANOZOOMER 2.0. compiled into a stack file after being aligned using ImageJ software. Stacks were projected and compiled into a single image with Imaris Scientific 3D/4D image processing and analysis software. Manual segmentation was performed for 3D reconstruction of total corpus callosum and hippocampal regions (Hippocampal formation and dorsal commissure). The thickness of the somatosensory cortex was measured on the same coronal sections stained with cresyl violet. We selected 3 to 9 non-consecutive slides at the level of the dorsal hippocampus, and measured cortex thickness on each side at a distance of 2 mm from the midline.

Scanning of ISH stains was done on the Nanozoomer 2.0 (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Using an original magnification of 40× the images were digitally analysed using the software Visiopharm Integrator System (VIS, Visiopharm, Hoersholm, Denmark), as we have described previously [35 (link)]. We performed a threshold analysis using an intensity interval of 0–180.

Spleen and kidney specimens were collected from euthanized animals on the days specified in each figure. Specimens were immediately fixed in 10% phosphate-buffered formalin upon removal, and then embedded in paraffin, sectioned transversely and stained with hematoxylin and eosin (H & E). Tissue imaging and analysis were performed with the NanoZoomer 2.0 from Hamamatsu Photonics K.K. (Hamamatsu, Japan). In some experiments, unstained paraffin sections were used for immunofluorescent staining. Paraffin sections on slides were treated with Target Retrieval Solution and Protein Block Serum-Free (Dako North America, Inc., Carpinteria, CA) according to the manufacturer’s protocol, and stained with respective primary and secondary antibodies with washing between and after in phosphate-buffered saline (PBS) with 0.1% Tween® 20. Resulting slides were briefly rinsed with PBS, desalted by dipping in distilled-deionized water, and sealed with coverslips in mounting medium with DAPI (Life Technologies Corporation, Grand Island, NY). A Zeiss LSM710 laser scanning confocal microscope (Carl Zeiss MicroImaging; Thornwood, NY) with EC Plan-Neofluar 10x/0.3, Plan-Apochromat 20x/0.8, and EC Plan-Neofluar 40x/0.75 objectives was used to scan the signals. Intensities of signals were also measured and shown as noted.

Bone marrowpecimens were immediately fixed in 10% phosphate-buffered formalin upon removal, and then embedded in paraffin, sectioned transversely and stained with hematoxylin and eosin (H&E). Tissue imaging and cell counts were performed with the NanoZoomer 2.0 from Hamamatsu Photonics K.K. (Hamamatsu, Japan) [4 (link)].