Ndp view2u12388 01

The fixed lung and tumor tissues were taken, dehydrated until transparent, and after embedding paraffin, cut it into 4 μm slices. The sections were stained with H and E. The slices were imaged using a NanoZomer digital slice scanner (Nikon, Tokyo, Japan), and the lung metastasis area was assessed using the NDP.view2U12388-01 digital pathology scanning software (Hamamatsu, Japan).

Animals were sacrificed by cervical dislocation. The testis and epididymis were dissected out en bloc, fixed in Bouin’s solution overnight, dehydrated in a graded ethanol series, and embedded in paraffin. Five-µm-thick serial sections with intervals of 50 µm were cut using a microtome and mounted on glass slides. Sections were treated with PAS-H to stain the basement membrane of seminiferous tubules, as previously described^{35 (link)}. Sections were digitized using a whole-slide scanner (Nanozoomer 2.0-HT C9600-13; Hamamatsu Photonics, Hamamatsu, Japan) with a 20-fold objective lens, and the resulting digital images of the sections were visualized with viewer software (NDP.view2 U12388-01; Hamamatsu Photonics).

Whole slide imaging (WSI) of entire sections was performed using a virtual slide scanner (Nano Zoomer-SQ, C13140-01, Hamamatsu Photonics K.K., Shizuoka, Japan). The settings for image capture were as follows: Object lens: 20× N.A. 0.75; scan mode: 40× mode; maximum capture size: 26 × 76 mm; pixel: 0.23 µm/pixel; light force: light-emitting diode; and image storage format: manual focus mode. We then randomly selected five fields of 40× WSI images as TIFF files using image-viewing software (NDP.view2; U12388-01, Hamamatsu Photonics K.K., Shizuoka, Japan).