Nanozoomer whole slide scanner

Antibiotic-treated mice were infected with a nonlethal dose of 1 × 10⁸ CFU by intranasal instillation, and at 18 h postinfection, the lungs were harvested and placed into 10% neutral buffered formalin (VWR) for 72 h. Tissue was processed by the NYU Experimental Pathology Research Laboratory. Tissues were processed into formalin-fixed paraffin-embedded (FFPE) tissue blocks using a Leica Peloris automated tissue processor. Five-micrometer paraffin sections were stained with hematoxylin and eosin (H&E) on a Leica BondRX autostainer, according to the manufacturer’s instructions. Slides were imaged on a Hamamatsu Nanozoomer whole-slide scanner.

Microscopic evaluation was carried out using the stained specimens that were previously scanned at twentyfold magnification with the aid of the Nanozoomer Whole Slide Scanner (Hamamatsu, Herrsching am Ammersee, Germany). All scanned slides were assessed by means of the NDP.view2 software (Hamamatsu Photonics, Hamamatsu City, Japan). Program Count Helper (Massako Sakanashi, Sakanapps, Japan) was used to help with manual cell counting. All tumor cells on the entire slide were evaluated with respect to the nuclear staining of each protein. Protein expression was expressed as the % of nuclear-stained tumor cells relative to all tumor cells on the slide. In accordance with the College of American Pathologists guidelines for immunohistology evaluation [16 (link),17 (link)], any nuclear tumor cell staining (even patchy) was taken as “no loss of expression” and only complete absence of nuclear staining was considered “loss of expression” provided that internal controls (e.g., keratinocytes, lymphocytes, and stromal cells) showed staining. Hence, MMR deficiency was considered when there was complete absence of nuclear staining for at least one protein. Cases with an MMR protein expression of less than the 10th percentile were classified as low-level MMR, and cases with an expression of more than the 10th percentile as high-level MMR.

After harvest and weighing, TA muscles were embedded in OCT and frozen in liquid nitrogen. The frozen muscles were sectioned perpendicular to their length and fibres were stained for laminin with a polyclonal rabbit anti-LAMA1 antibody (1:2,000, Sigma-Aldrich). Slides were scanned by Aperio Scanscope AT (Leica Microsystems Inc.). Fibre number and cross-sectional area were analysed by HALO software (Indica Labs). Fibre size is represented as average size of the total number of fibres in a cross-section of TA muscle.
Monkey muscle specimens were fixed in 10% neutral buffered formalin and transferred within 48 h to 70% ethanol. After fixation, the muscles were trimmed to obtain a maximum number of fibres in cross-section. Trimmed tissues were processed into paraffin blocks, sectioned at 4–6 μm, mounted on glass microscope slides and stained for dystrophin to outline the sarcolemma and allow for automated image analysis. Slides were scanned using the Hamamatsu NanoZoomer whole slide scanner and images were imported into the Visiopharm software platform. Image analysis protocols were created to count individual muscle fibres and the cross-sectional area was determined for each fibre using automated image analysis64 (link).