Nanozoomer 2.0 rs system

Expression level of PCDH8 was determined based on the immunohistochemistry protocol (Paraffin) from Cell Signaling Technology (Beverly, MA) as previously described.21 (link) Immunohistochemical analysis was performed using PCDH8 antibodies (1:100 dilution, Abcam, Cambridge, MA) and secondary antibody conjugated with HRP (SuperPicture Polymer Detection Kit, HRP, broad spectrum; Thermo Fisher Scientific, Inc., Waltham, MA).
Assessment of PCDH8 staining was conducted according to staining showing and positive cells percentage. Thereby, PCDH8 staining patterns were categorized into 5 groups: 1+, less than 10% of cells stained positive; 2+, 10% to 25% positive cells; 3+, 26% to 50% positive cells; 4+, 51% to 75% positive cells; 5+, greater than 75% positive cells. The PCDH8 staining images for IHC and hematoxylin-eosin staining were obtained using the NanoZoomer 2.0-RS system (Hamamatsu Photonics Inc., Germany), and the digital slides were analyzed by NDP. view 2.5.14 version.

IHC was executed as previously described.23 (link) The antibodies used in the IHC were anti-KDM5A and anti-ki-67 (Abcam, USA). The KDM5A protein would be quantified with the proportion and intensity of positively stained cells across three randomized images. We used the NanoZoomer 2.0-RS system (Hamamatsu Photonics Inc., Germany) to obtain the images and the software NDP.view 2.5.14 version to analyze the digital slides.

IHC was performed as described previously.19 (link) Briefly, TMA sections were deparaffinized, rehydrated and underwent antigen retrieval. They were then blocked for an hour at room temperature. After incubation with primary antibody CD44 (dilution 1:100, Proteintech Group, Wuhan, China) at 4°C overnight, the slide was probed with biotinylated goat anti-rabbit secondary antibody and then detected by SignalStain^® DAB (CST, Danvers, MA, USA) and counterstained with Hematoxylin QS (Vector Laboratories, Burlingame, CA, USA). The images of IHC and H&E staining were obtained using the NanoZoomer 2.0-RS system (Hamamatsu Photonics Inc., Herrsching, Germany), and the digital slides were analyzed by the software of the NDP.view 2.5.14 version. Two pathologists (Xiaolong Chen and Yan Yu) scored the IHC simples in a blinded manner independently. Sections were semi-quantitatively scored for the CD44 staining patterns as follows: the staining extent in each core was scored as 1+ (<25% staining of tumor cells), 2+ (25%–50% staining of tumor cells), 3+ (50%–75% staining of tumor cells) or 4+ (>75% staining of tumor cells), who were blinded to the clinicopathological data, with a consensus reached in all cases. Scores of 3+ and 4+ were defined as exhibiting high expression and scores of 1+ and 2+ were deemed as exhibiting low expression.