Nanozoomer 2.0 scanner

Ears from 8- to 10-week-old mice were collected and fixed in 4% paraformaldehyde overnight at 4°C. For each ear, the dorsal skin was separated from the rest of the tissue (central cartilage and the adhering ventral skin). Whole dorsal ear skins were fixed in methanol for 1 hour at –20°C and then blocked with 3% milk and 0.2% Triton X-100 for 1 hour at room temperature. They were then incubated with a polyclonal goat anti-LYVE1 antibody (1:200; AF2125, R&D Systems) and rat anti-CD31 antibody (1:150, DIA-310, Dianova) overnight at room temperature. After washing in PBS, an Alexa Fluor 488–coupled rabbit anti-goat antibody (1:200; A21222, Invitrogen) was added for 2 hours at room temperature. After washes with PBS, Alexa Fluor 546–coupled goat anti rat antibody (1:200; A11081, Invitrogen) was incubated at room temperature for 2 hours. Slides were then washed with PBS and mounted using Dako fluorescent mounting medium (Dako). Slides were scanned using a Nanozoomer 2.0 scanner (Hamamatsu) and visualized using NDPviewer (Hamamatsu). The lymphatic vessel frequency, density, and thickness were quantified using ImageJ software.

Archival BMBs from 34 MM patients diagnosed at the Unit of Pathology, A.O. San Paolo, Department of Health Sciences, University of Milan, Italy were evaluated in the present study approved by the Ethical Committee of Milano University (No. 8/15—4 March 2015).
Histopathological diagnosis of MM was carried out according to the WHO classification criteria; BMPs were subdivided according to the extent of the BM infiltration by the myeloma cells as follows: degree of infiltration I = less than 20% (10 cases); II = 21–50% (11 cases), III > 51% (13 cases). Notch activation and angiogenesis were investigated by immunohistochemistry (IHC) with an automatic immunostainer (DAKO OMNIS) using diaminobenzidine as the chromogen. Used antibodies were reported in Table 3. Digital images were obtained by the NanoZoomer 2.0 scanner (Hamamatsu Photonics, Japan).
Statistical analyses were performed using GraphPad Prism 6 software (GraphPad software, San Diego, CA, USA). For the in vitro assays including two groups, we carried out one-tailed Student’s t-tests; when including 3 or more groups, we performed a one-way ANOVA with Tukey’s post-hoc tests. For in vivo experiments, the minimum size of each group was determined using an a priori power analysis for a one-way ANOVA with an alpha = 0.05 with G-power 3.2 software.

Thermal cauterization was induced on the corneas of 8 to 10-week-old mice. After 7 days, corneas were dissected and whole mounted for immunostaining as previously described (28 (link)–30 (link)) or snap frozen for RNA extraction. To visualize lymphatic and blood vessels, corneas were fixed in methanol at –20°C for 1 hour and then blocked with 3% milk for 1 hour at room temperature. Corneas were then incubated overnight at room temperature with a polyclonal goat anti-mouse LYVE1 antibody (1:200; AF2125, R&D Systems) and with a monoclonal rat anti-mouse CD31 (1:200; 553370, BD Biosciences). After washing in PBS, an Alexa Fluor 488–coupled rabbit anti-goat antibody (1:200; A21222, Invitrogen) was added for 2 hours at room temperature. After washing in PBS, an Alexa Fluor 546–coupled goat anti-rat antibody (1:200; A-11081, Thermo Fisher Scientific) was added for 2 hours at room temperature. After washing, the slides were scanned using a Nanozoomer 2.0 scanner (Hamamatsu) and visualized using NDPviewer (Hamamatsu). The lymphangiogenic responses were analyzed using a described computerized method (28 (link), 31 (link)). All the results were normalized to the total cornea area and are expressed as a percentage of WT control.