Nanozoomer s60 slide scanner

All the mice infected with a CFU of 2000 or below were weighed and sacrificed on day 30 post-inoculation. We referred to a previous study and developed a clinical score method to calculate the severity of disease progression [26 (link)]. The mice were scored in terms of general behavior, feeding habits, and weight gain or loss. Animals reaching a clinical score of ≥4.0 were at the time point that followed by the time of death. The weight of each lung and spleen was measured, and organ coefficient was obtained by using the following formula: organ coefficient = organ weight (g)/mouse weight (g) [27 (link)]. Blood was obtained ante-mortem from eye route and serum was collected after clotting. The blood serum samples were stored at −80 °C until further experiment. The same lobe of the lung from each study group was fixed in 4% phosphate-buffered formaldehyde and embedded in paraffin. Sections of ~5 μm thickness were prepared and stained with haematoxylin and eosin (H&E), and with Ziehl-Neelsen (ZN) for histological examination and for the detection of acid-fast bacilli, respectively [28 (link)]. The digital images of the whole slide were acquired by Hamamatsu NanoZoomer S60 slide scanner and its viewing platform (NDP Viewer).

Right lungs were inflated with 4% buffered formalin to 25 cm of fixative pressure. Non-inflated left lungs were immersion fixed. Lungs were paraffin embedded and sectioned at 5 µm and stained with H&E, Masson-Goldner-Trichrome and AB-PAS. Images were captured with a NanoZoomer S60 Slidescanner (Hamamatsu, Hamamatsu City, Japan) at a magnification of 40×. Airway regions were determined from proximal-to-distal distances and airway branching, as determined by longitudinal sections of lung lobes at the level of the main axial airway, as previously described [55 (link)]. Degenerative cells were identified by morphologic criteria such as swollen cells with vacuolized cytoplasm and pycnotic nucleus in H&E stained lung sections. Numeric cell densities were determined using NDP.view2 software version 2.7.52 (Hamamatsu, Hamamatsu City, Japan), as previously described [16 (link)].

Frozen tissues were embedded in optimal cutting temperature (OCT) compound and cryo-sectioned at 25 μm on PEN-membrane slides and fixed in 70% ethanol for 5 min. Fixed tissue sections were rinsed twice with 1X PBS, stained with haematoxylin and eosin (H&E), and photo-recorded using a Nanozoomer S60 Slide scanner (Hamamatsu). Crypts of interest were marked using the NDP.View2 software (Hamamatsu) for subsequent laser-capture microdissection (LCM).