Neg 50tm frozen section medium

We immuno-stained 10 µm-thick sections of the sample obtained with a cryostat (Thermofisher, Cryostar nx70). After imaging, samples were fixed in 4% formaldehyde, incubated overnight in 30% sucrose and immersed in pre-warmed embedding solution (7.5% gelatin and 10% sucrose in DI water). Blocks containing multiple organoids were cut out and frozen in − 50 °C isopentane bath. With confocal microscopy (LS980 Zeiss, 40x) we could detect the presence of SOX-2 (ThermoFisher, Invitrogen, eBioscience™, 14-9811-82) and Nestin (Abcam, ab81462) (Fig. 3, Suppl. Fig. S2) within the structures. For TUBB3 (Fig. 3, Suppl. Fig. S2) and N-CAD (Suppl. Fig. S2) staining, samples were embedded and cryosectioned directly in Neg-50TM Frozen Section Medium (Thermo Fisher Scientific, 6502B) and stained with anti-N-Cadherin antibody (ThermoFisher, Invitrogen, PA5-29570) and anti-Tubulin ß 3 antibody (Clone Tuj1, BioLegend, MMS-435P) respectively.

For further proof of correct drug application, tissue samples (thigh muscle) were taken from anatomical regions proximal to the cannulation site and from the contralateral limb to exclude leakage and systemic dispersion. As a positive control, three samples were obtained from the perfused limb. These samples were immerged in 4% PFA in PBS (pH 7.4) for 16 days, then washed in PBS three times for 30 min, and immersed in EDTA decalcification solution for 90 min. Then, samples were embedded in Neg-50^TM frozen section medium (Thermo Fisher Scientific, Waltham, MA, USA). Finally, samples were cryosectioned and analyzed by fluorescence microscopy with Axiovert 200 M (Zeiss, Oberkochen, Germany). Microscopic analysis enabled the visualization of doxorubicin via fluorescence after excitation at 560 nm (100 ms, mCherry mode) with DAPI counterstain (20 ms, DAPI mode) at 40× magnification. Magnification was further increased to 200× utilizing ImageJ (LOCI, University of Wisconsin, Madison, WI, USA).