Nanozoomer digital slide scanner

Formalin-fixed, paraffin-embedded mouse livers were cut and stained with hematoxylin and eosin (H&E) or Picrosirius Red (Abcam) dyes. Liver histology was blindly scored by an independent histopathologist based on three semiquantitative items: steatosis (0–3), lobular inflammation (0–3) and hepatocellular ballooning (0–2) (not shown) (Liang et al., 2014 (link)). Stained sections were scanned with NanoZoomer Digital slide scanner (Hamamatsu) and quantification of Picrosirius Red-stained areas was performed using ImageJ on three independent areas per section. Data is represented as the average positively-stained percent of area of interest.
Hepatic macrophage content was assessed by quantification of F4/80 positively-stained areas (ab6640, Abcam).

Knees were decalcified in 5% formic acid (Decal Chemical Corp.) and processed for standard paraffin embedding. Five-micrometer thick serial sagittal sections were cut extending through the entire joint width. Every 10^th section was stained with Safranin-O to evaluate cartilage proteoglycan7 (link)9 (link). The cover-slipped sections were imaged using NanoZoomer digital slide scanner (Hamamatsu) and cartilage injury was identified. Using three selected Safranin-O stained sections synovitis was evaluated as before25 (link).

Each 2-mm plaque segment was cryo-sectioned into four 500-μm blocks in line with the SPECT resolution. Within each 500-μm block, sequential adjacent sections of 5 μm were made which overall yielded a total of 262 sections for quantification and analysis with respect to the SPECT/CT scan data. Sections were immunohistochemically stained with anti-LFA-1 (mouse anti-human CD11a 1:100 Biorad, MCA1848 clone 38), and anti-SST_2A (human tissue: rabbit anti-human SSTR2A 1:50, clone UMB1, Abcam). Haematoxylin and eosin staining was used to determine the plaques overall structural morphology. The slides were photographed using a NanoZoomer Digital slide scanner (Hamamatsu, Photonics K.K). All images were exported to BioPix iQ 3.3.5 software for quantitative analysis. The areas of positively stained LFA-1 and SST₂ were quantified using the dedicated hue, saturation and brightness selection tool, and positive areas were measured as a percent fraction of the total plaque tissue area.

For cryosectioning, eyes were enucleated and fixed with 4% paraformaldehyde in PBS or FAS eyeball fixative solution (G1109, Servicebio, Beijing, China), the lens and cornea were removed, and the eye cups were embedded in optical cutting temperature compound (OCT, 4583, Tissue-Tek, Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. The eye cup was sectioned to a thickness of 7 μm using a cryostat. For histological analysis, sections were collected at regular intervals from approximately 24 sites per eye. Sections were stored at –80°C and used within 2–3 days.
Before use, the cryosections were fixed on slides for 10 min with acetone. The sections were then washed thrice with PBS, followed by IF staining. The primary antibodies used were monoclonal mouse 4D2 antibody and biotinylated peanut agglutinin (PNA; 1:200 dilution, B-1075, Vector, CA, USA). The secondary antibodies used were donkey anti-mouse conjugated with Alexa Fluor 568 for 4D2, and rhodamine (TRITC)-conjugated streptavidin (1:200 dilution, 123126, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for PNA. 4D2 and PNA were used to identify the rod and cone photoreceptors, respectively. Cryosections were also stained with HE, and whole slide images were obtained using a NanoZoomer digital slide scanner (Hamamatsu Photonics, Hamamatsu City, Japan).

Upon termination, the hearts from the rats with MR that were terminated at 2 and 10 weeks, and the age- and weight-matched sham rats (n = 3/group) were perfusion fixed with 10% formalin. A 16-G needle was inserted into the aorta, with its tip placed above the aortic valve, and formalin was injected with a syringe to perfuse the myocardium through the coronary arteries. The hearts were left in 10% formalin for 24 h, and then transferred to 70% ethanol. Histopathology was performed at an independent site, Alizee Pathology (Thurmont, Maryland), where tissue sectioning, processing, paraffin embedding, hematoxylin, and eosin staining and Gomori's elastin trichrome staining were performed. Slides were imaged with the NanoZoomer digital slide scanner (Hamamatsu, C13210-01) at the Winship Pathology Core at Emory University. From each cross-section of the hearts in each group, the cardiomyocyte cross-sectional area was measured using ImageJ software (NIH, USA). Individual cells were identified, and their border was traced and the pixels within the border were calibrated against a scale to measure the area. In each image, cardiomyocyte cross-sectional area was measured from 20 or more different cells, and the data were averaged.

Following paraformaldehyde fixation, biopsies of 8 sites from each control (n = 9) and FGR (n = 7) placenta were embedded in paraffin using routine histological techniques and sectioned at 7 µm thickness. Sections were rehydrated using xylene and ethanol gradients, stained with hematoxylin and eosin, dehydrated with ethanol gradients and xylene, then mounted with DPX. Slides were then scanned using a Nanozoomer digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan).