Hematoxylin

Formalin-fixed paraffin-embedded mouse spleens were cut into 10 μm sections. The tissue slides were deparaffinized, rehydrated in decreasing alcohol concentrations and incubated in 3% H₂O₂ for 10 min to block endogenous peroxidase activity. Antigen retrieval was performed at 98°C for 50 min in 10 mM HIER citrate buffer pH 6 (Zytomed Systems, Berlin, Germany) in the water bath. Nonspecific protein binding was blocked with 2% goat serum and 2% BSA in PBS for 30 min. The slides were then incubated with anti-STAT5 antibody (1:200, Santa Cruz Biotechnology) overnight at 4°C. After incubation for one hour with the second anti-rabbit antibody (EnVision+ System-HRP labelled Polymer, DAKO, Hamburg, Germany) the signal was visualized by incubating the slides with AEC substrate (DAKO) and the nuclei were counterstained with hematoxylin (Carl Roth, Karlsruhe, Germany). Spleen sections from mice with FLT3-ITD-driven leukemia or transplanted with mock-transduced HSPCs were used as positive and negative controls, respectively. Staining with the secondary antibody served as further negative control

Pancreatic sections (2 µm) were de-paraffinized and rehydrated in Roti-Histol (Carl Roth, Karlsruhe, Germany) and a decreasing serial solution of ethanol. Slides were heated in citrate buffer (10 mM citrate acid, 0.05% Tween 20) in a pressure cooker using a microwave for 30 min at 900 W followed by a cooling step (30 min, RT). Sections were incubated with blocking solution (5% BSA/TBST, 1 h, RT), followed by primary antibodies (1 h, RT). Rabbit anti-insulin (Abcam, Cambridge, UK, ab181547) was used as primary antibody. Hydrogen peroxide blocking was performed with a 0.3% solution (Carl Roth) for 15 min at RT, followed by incubation with HRP-labeled Goat Anti-Rabbit IgG (1 h, RT) (Abcam, ab205718). Before mounting with VectaMount Mounting Medium (H-5000), tissue sections were incubated with 3,3’-diaminobenzidine in chromogen solution (ImPACT DAB Peroxidase Substrate Kit, Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin (Carl Roth). Stained insulin in whole pancreatic sections were imaged with a MIRAX-MIDI Scanner (Carl Zeiss AG, Oberkochen, Germany), equally edited with Pannoramic Viewer and analyzed with ImageJ Software.

For histological analysis, fixed embryos were rinsed in PBS and dehydrated using ethanol. They were embedded in paraffin and sectioned transversally at 7 µm thickness.
Paraffin sections were deparaffinized with RotiHistol (Carl Roth, Karlsruhe, Germany) and rehydrated for staining.
For hematoxlin-eosin (HE) staining the sections were stained with hematoxylin for 15 min and eosin for 2 min (Carl Roth, Karlsruhe, Germany).
Masson-Goldner-Trichrome-Staining was used in order to differentiate the connective tissue. First, the nuclei were stained for 5 min using hematoxylin according to Weigert. Then, the trichromatic stain was performed using ponceau-acid fuchsin, phosphotungstic acid-orange G, and 0.2% light green (Carl Roth, Karlsruhe, Germany). For differentiation, 1% acetic acid was used.
After both staining procedures, the sections were dehydrated again and covered. The sections were analyzed microscopically and photographed using the virtual slide microscope VS120 (Olympus, Tokyo, Japan). Histological measurements and cell density calculation were performed with the OlyVia software (Version 2.9, Olympus, Tokyo, Japan) and QuPath (Open source software, Version 0.3.2). The beads were indicated by circles in order to improve visualization.

The OTCs and foreskin were embedded in tissue freezing medium (Jung, Nussloch, Germany), and snap-frozen with 2-methylbutan (Roth, Karlsruhe, Germany) on dry ice before preserving at -80°C. Cryostat sections of 4 μm thickness were mounted on glass slides, dried for 2 hours at room temperature (RT), stained with hematoxylin (Roth, Karlsruhe, Germany) and eosin (Sigma Aldrich) (H&E), and monitored using an AX70 microscope (Olympus, Hamburg, Germany).

Formalin fixed and paraffin embedded tissue sections (3 µm) were stained using the VECTA Stain Elite Kit (Vecta Laboratories, Burlingame, USA) according to manufacturer’s instructions. After deparaffinization at 60 °C for 45 min, sections were rehydrated using descending alcohol concentrations. Demasking of epitopes was achieved by boiling in citrate buffer (10 mM trisodium citrate dihydrate, pH 6). Endogenous peroxidase was blocked by incubating in 3% H2O2 in methanol for 30 min before incubation in 1.5% goat serum for blocking of unspecific binding. Followed by incubation with primary antibody (anti-CAPN1, Abcam ab39170, 1:200; anti-CAPN2, Abcam ab39165, 1:100; diluted in PBS) at 4 °C overnight. Incubation with the respective secondary biotinylated antibody and ABC reagent were followed by DAB staining with the ImmPACTT DAB Kit (Vecta Laboratories, Burlingame, USA). For counterstaining, hematoxylin (Carl Roth, Karlsruhe, Germany) was used. Finally, sections were dehydrated by ascending ethanol concentrations and covered with mounting medium. Images were acquired using a Leica Axiophot XX with an integrated camera system.

For differentiation, pediatric LR-MSCs (passage 5) and ovine LR-MSCs (passage 3) were plated in a 24-well plate in regular culture medium and placed in a humidified atmosphere at 37°C with 5% CO₂. After 48 hours, cells were washed with PBS and complete differentiation media of the different StemPro Differentiation Kits (Thermofisher) was added. For adipogenic induction cells were plated at 10⁴ cells/cm², for chondrogenic induction cells were plated at 2.7x10⁴ cells/cm², and to induce osteogenic differentiation, cells were plated at 5x10³ cells/cm². Medium was changed twice a week and after 15–21 days, cells were fixed with 4% PFA (Sigma). Adipocytes were stained with Oil Red O (Sigma) to detect the formation of lipid droplets and counterstained with hematoxylin (Carl Roth). Chondrocytes were stained with Alcian Blue (Sigma) to visualize glycosaminoglycan synthesis and Alizarin Red S staining (Sigma) was used to detect calcium deposits in osteocytes.

Mouse lung tissues were fixed using 4% PFA followed by embedding in paraffin. Paraffin blocks were sectioned into 5-μm-thick slices and placed on glass slides. Following deparaffinization, lung sections were stained with hematoxylin (Roth) for 2 min, washed with running tap water for 10 min, and then stained with eosin (Thermo Fisher Scientific) for 2 min.