Nx70 cryostat

Half of the SKOV-3 tumors were harvested and frozen in a cryoprotective gel (Tissue-Tek optimal cutting temperature compound, Sakura) using dry ice for autoradiography and histology. They were then cut using the NX70 cryostat (Thermo Fisher Scientific) set at a temperature of -15 ºC, mounted on Superfrost Plus Gold slides and fixed in methanol for 5 min at room temperature. For autoradiography, a phosphor screen was applied on 14 μm-thick sections and the resulting signals were acquired with the PhosphoImager (GE Typhoon FLA 9500). The consecutive sections of 8 µm were stained with Hematoxylin and Eosin (H&E) following supplier recommendations (Leica Biosystem).

Organoids were fixed in 4% paraformaldehyde (PFA) for 1 h at RT and washed with phosphate‐buffered saline (PBS) three times for 10 min each at RT before allowing to sink in 30% sucrose at 4℃ overnight and then embedded in OCT (Agar Scientific, cat. #AGR1180) and cryosectioned at 14 μm with a Thermo Scientific NX70 Cryostat. Tissue sections were used for immunofluorescence and for the senescence‐associated β‐galactosidase assay. For immunofluorescence, sections were blocked and permeabilized in 0.1% Triton X‐100 and 3% bovine serum albumin (BSA) in PBS. Sections were incubated with primary antibodies overnight at 4℃, washed and incubated with secondary antibodies for 60 min at RT. 0.5 μg ml⁻¹ DAPI (Sigma, cat. #D9564) was added to secondary antibody to mark nuclei. Secondary antibodies labelled with Alexafluor 488, 568 or 647 (Invitrogen) were used for detection.

Following the aforementioned tissue co-incubations overnight, aortic rings were immediately washed with PSS, and subsequently stabilised in fresh PSS, at 37°C for 60 min. Next, rings were fixed in optical cutting temperature compound (OCT; Tissue-Tek® O.C.T.™) and immediately snap-frozen at -20°C. Rings were cryosectioned (8 μm) and collected using an NX70 cryostat (Thermo Scientific Inc., Runcorn, UK) and mounted on histological slides in duplicate. For each ring, one section was incubated with Dihydroethidium (DHE, 5 μM; Sigma-Aldrich, Germany) and another with PSS solution (control) in darkness for 15 min. After incubation, the sections were washed with deionized H₂O twice (1 min). Following excitation by a xenon lamp, images (x10 magnification) were immediately captured using a Nikon fluorescence microscope (TE2000-U, Japan) at an emission wavelength of 570–645 nm (TRITC Filter), using a digital microscope camera (Nikon DS-5M, Japan) and quantified by ImageJ 2.0 software. The DHE-derived 2-OH-E+ levels (A.U) represent the difference between the incubation with PSS and DHE.

Mice were anesthetized by an intraperitoneal injection of 20% (w/v) sodium pentobarbital (Pentoject; Animalcare, York, UK): 0.01 ml for P1–P7, 0.05 ml for P14–P21, 0.1 ml for P28-P56. After complete anesthesia, phosphate-buffered saline (PBS; Oxoid, Basingstoke, UK) at 5 ml for P1–P14 and 10 ml for P21-P56 was perfused transcardially, followed by 4% (v/v) paraformaldehyde (PFA; Alfa Aesar, Heysham, UK) at 5 ml for P1–P14 and 10 ml for P21–P56. Whole brains were dissected out and immediately postfixed at 4 °C in 4% PFA (2 h for P1–P14, 4 h for P21–P56) before transfer into 30% (w/v) sucrose (VWR Chemicals, Lutterworth, UK) at 4 °C in 1×PBS. Brains were then embedded into Optimal Cutting Temperature (OCT; CellPath, Newtown, UK) medium within a cryomould and frozen in isopentane cooled with liquid nitrogen. Brains were then sectioned in the parasagittal plane at 18 μm thickness using an NX70 cryostat (Thermo Fisher Scientific, Gloucester, UK). Cryosections were mounted on Superfrost Plus glass slides (Thermo Fisher Scientific) and stored at −80 °C.

BVOs were rinsed twice in PBS and then fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT) and washed twice in PBS. BVOs were dehydrated in 20% sucrose at 4 °C overnight, then embedded in Gelatin solution and stored at −20 °C for cryosectioning. The frozen BVOs were sectioned using NX70 Cryostat (Thermo Scientific) to 20 and/or 70-μm thickness. Frozen sections were then washed in PBS for 5 min and then permeabilized and blocked in Blocking buffer for 2 h at RT. Primary antibodies were diluted in Blocking buffer and incubated overnight in a cold room. Antibodies used and dilution factors’ information are available in Supplementary Table S1. On the following day, sections were washed three times in PBS with 0.1% TritonX-100 (PBS-T) and then incubated with labelled secondary antibodies for 2 h at RT. After two washes in PBS-T, the sections were counterstained with DAPI and mounted with Fluoromount-G mounting media (Thermo, 00-4958-02).
For immunostaining intact BVOs and VNs were rinsed in PBS then fixed in 4% PFA for 30 min at RT and washed twice in PBS. The fixed VNs were stored in PBS at 4 °C for up to a month. Stained BVOs were mounted into iSpacer (0.5 mm deep imaging Spacer, SunJin Lab). Samples were viewed and imaged using the Spinning Disk Confocal System (Nikon) and the Operetta CLS High-Content Analysis System (PerkinElmer) at a 20x or 40x magnification.