Ndp view2 viewing software

Fixation, paraffin-embedding, and sectioning of tumor samples were performed by the histopathology core service at the Centre for Genomics and Oncological Research (Granada, Spain). In order to evaluate the percentage of proliferating cells, tumor sections were immunostained with Ki67 and counterstained with hematoxylin and eosin (H&E) at Atrys Health (Barcelona, Spain). The staining was visualized using NDP.view2 Viewing software (Hamamatsu), and Ki67 coverage was quantified on ten different images per tumor using Fiji software. In order to measure BG4 signal, tumor sections were dewaxed and rehydrated following standard methods. Epitope retrieval was performed at 100 °C for 20 min with citrate buffer (citrate-based buffer pH 6.0) according to previous studies [13 (link)]. After blocking, staining was achieved with BG4 antibody overnight at 4 °C, following a 1 h incubation with anti-FLAG at RT and a 30 min incubation with an anti-mouse antibody at RT in darkness. Slides were then counterstained for 5 min with DAPI to visualize the cell nuclei. Antifade Mowiol (81381, Merck, Darmstadt, Germany) was used as mounting media. Images were acquired on a Confocal Zeiss LSM 710 inverted microscope with a 63× immersion objective. BG4 mean nuclear fluorescence intensity was quantified using Fiji software (N > 2000). Antibodies used are listed in Supplementary Table S1.

The tumor tissues were fixed with 10% neutral formalin, embedded in paraffin, and then sliced into 4 μm thick paraffin sections. Dewaxed sections were stained with hematoxylin and eosin (HE). Without an integrated cell structure, the necrotic area was stained red under a light microscope. NDP.view2 Viewing software (Hamamatsu Photonics, Shizuoka, Japan) was used to quantify the red necrotic area percentage. These analyses were all conducted in a blinded manner by two experienced pathologists.

4% PFA-fixed paraffin-embedded tissues were sectioned at 5 μm and mounted onto Apex Superior Adhesive Slides (Leica, Wetzlar, Germany). Standard deparaffinization was done in xylene and serial alcohol immersion. Heat-induced epitope retrieval was performed in eBioscience IHC Antigen Retrieval Solution, pH 6.0 (Thermo Scientific) by pressure cooking at 125°C for 30 s and gradual cooling to 90°C over 40 min. Slides were stained using Dako Autostainer Plus (Dako Omnis; Agilent, Santa Clara, CA, United States) slides were rinsed with buffer and blocked with H₂O₂ block for 10-min, Dako envision kit Protein Block (Dako Omnis). Slides were incubated with S100A9 primary antibody (1:3000 dilution, NB110–89726; Novus Biologicals) for 1 h with a posterior 30-min Dako Rabbit Labeled Polymer secondary antibody (Dako Omnis), washed (×2) for 7-min with Dako DAB + Chromogen (Dako Omnis), and counterstaining with blue Mayer’s Hematoxylin (Sigma Aldrich). Slides were imaged and scanned using a NanoZoomer 2.0-HT. Images were analyzed by NDP.view2 Viewing software (Hamamatsu Photonics, Hamamatsu, Japan) and ImageJ.

Due to the limited penetration of the X-gal substrate in larger tissues, X-gal staining was also performed directly on cryosections for time points over E13.5, and for all adult and neonatal MI hearts. Dissected hearts were fixed for 1–3 h, depending on size, in 4% PFA in PBS at 4 °C, then incubated overnight in 30% sucrose at 4 °C. They were then washed in a 50/50 mix of 30% sucrose/OCT Embedding Medium (Thermo Scientific), followed by a minimum of two washes in OCT before mounting over dry ice and storing at −80 °C.
Cryosections were cut at a thickness of 15 μm, and allowed to thaw at room temperature before washing in PBS to remove the OCT embedding medium. Sections were incubated in Fix solution (4% PFA, 2 mM MgCl₂, 5 mM EGTA in PBS) for 10 min at room temperature, before further washes in PBS. They were then incubated in Cryosection Staining solution (2 mM MgCl₂, 0.02% Nonidet P40, 0.01% sodium deoxycholate, 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, and 1 mg/ml X-gal in PBS) in a humidified chamber at room temperature for 1 h to overnight, depending on staining intensity.
After staining, sections were washed in PBS, fixed in 4% PFA in PBS for 15 min, counter-stained with Nuclear Fast Red and imaged using a NanoZoomer S210 slide scanner with NDP.view2 viewing software (Hamamatsu).

After trimming the epoxy resin-embedded tissue, ultrathin sections 70 nm thick were cut using an ultramicrotome (EM UC 6; Leica, Wetzlar, Germany), mounted on grids, and stained with 4% uranyl acetate and lead citrate for TEM using an H-7650 or an HT7700 (Hitachi, Tokyo, Japan). For SEM, ultrathin sections (200 nm) were cut using an ultramicrotome fitted with a histo-Jumbo diamond knife (DiAtome, Biel, Switzerland). Serial sections were placed on platinum-palladium-coated (MC1000; Hitachi, Tokyo, Japan) glass slides and allowed to adhere at 55°C for 30 min. The sections were stained with 1% toluidine blue for light microscopy and scanned with a NanoZoomer 2.0-RS digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan). The observation regions were selected using NDP.view2 viewing software (Hamamatsu Photonics, Shizuoka, Japan). The sections were stained with 1% uranyl acetate and lead citrate and observed by ultra-high-resolution field emission SEM (SU8010; Hitachi, Tokyo, Japan). Regions of the ultrathin sections were imaged using backscattered electrons with an accelerating voltage of 1.5 kV. Images were taken at magnifications of ×7,000 to ×9,000, with a working distance of 3 mm. The workflows are shown in Fig. 3.