Eosin g

For histological analysis mice were sacrificed at the indicated time, kidneys were collected, weighted and fixed in formalin 10% (BioOptica, #05-01005Q), included in paraffin and cut 5 μm/slides. Kidney sections were air-dried and rehydrated in PBS (Sigma-Aldrich, #P4417). For YAP staining kidney sections were incubated with YAP antibody and 5 min in Eosin G (BioOptica, #05-10002/L); For Ki67 staining kidney sections were incubated with Ki67 antibody and 2 min in Hematoxylin (BioOptica, #05-06015/L). Sections were then washed, processed through a dehydration alcohol scale and mounted in DPX (Sigma-Aldrich, #06522). For the semi-automated quantification, slides were acquired with Aperio AT2 digital scanner at magnification of 40 × (Leica Biosystems) and analyzed with Imagescope (Leica Biosystem). YAP and Ki67 nuclear staining per cyst was performed by Aperio Image analysis software (Leica Biosystems) and counted manually.

3D bioprinted scaffolds containing CLL primary cells after 7 days of culture were washed twice with HBSS with 50mM of CaCl₂ for 8 minutes at 37°C, and then fixed with 4% PFA containing 50mM of CaCl₂ o/n at 4C. After fixation, scaffolds were washed twice with HBSS with 50mM of CaCl₂ for 10 minutes RT and eventually incubated, first, for 45 minutes at 4°C in HBSS with 50mM, then for another 45 minutes RT in sucrose 30% in PBS. The scaffolds were then embedded in OCT matrix and placed at -80°C until cryosectioning. Frozen samples were sectioned (5-7µm) on Superfrost-plus microscope slides (Thermo Fisher Scientific, Massachusetts, USA), washed one time with PBS, and then stained with Mayer’s Hematoxylin (Bio-Optica, Milan, Italy) for 1 minute, washed with tap water and stained with Eosin G (Bio-Optica, Milan, Italy) for 2 minutes. Images were taken with Zeiss Axio Imager M2m microscope with AxioVision (Rel. 4.9.1) software, and then processed using FIJI (ImageJ) software. Images of MEC-GFP cells inside and outside the 3D bioprinted scaffolds were obtained by using JuLI™ Stage fluorescent microscope.