Cm3050s cryomicrotome

Tumors were embedded into a 2% CMC, 1% gelatin hydrogel for simultaneous processing. Fresh frozen tissues were cryosectioned to 10 µm thickness using a Leica CM3050S cryomicrotome (Wetzlar, Germany). Data were recorded using a desorption electrospray ionization mass spectrometry imaging (DESI-MSI) system consisting of a Omnispray 2D DESI source (Prosolia Inc, Indianapolis, IN, USA) and a Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with a home-built sprayer as described previously 34 , 35 (link). Methanol/Water (95:5 v/v) was used as electrospray solvent at a flow rate of 1.0 µL/min delivered using a Dionex Ultimate 3000 nLC pump (Sunnyvale, CA, USA). Data was recorded using a spatial resolution of 50 µm in x-direction and 75 µm in y-direction, negative ion mode and using a mass range of m/z 250-1000. More details can be found in Table S4. Imaging data were converted into mzML format using MSIConvert tool from the ProteoWizard 3.0.4043 toolbox 36 (link) and subsequently converted into .imzML format using imzMLConverter v1.3 37 (link).
Drug detected is always total drug, in this manuscript AZD2811 is total drug detected which was dosed as NP-formulation (i.e. encapsulated plus released AZD2811), while [²H₅]-AZD2811 is total drug detected which was dosed as free drug.

DEHP-treated testis were fixed 4% paraformaldehyde for overnight at 4°C and embedded in Optimum Cutting Temperature (O.C.T.) compound (Sakura Finetek 4583). The embedded samples were sectioned using a CM3050S cryomicrotome (Leica) to a thickness of 10 μm. The sections were permeabilized, blocked, incubated with Anti-Cleaved Caspase-3 (CST 9661, RRID:AB_2341188, 1:400 dilution) for overnight at 4°C, and then incubated with Anti-rabbit Alexa 488 (Invitrogen A21206, 1:500 dilution) and 1 μg/mL DAPI for 1 hr at room temperature. The sections were washed after primary and secondary antibody treatments. Samples were mounted using Vectashield (Vector H-1000) and observed under a TCS SP8 confocal laser scanning microscope (Leica).

Sudan Black B staining (SBB staining) was performed using a previously published protocol (Georgakopoulou et al., 2013 (link)). In short, OCT-embedded and snap-frozen tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome. Frozen sections were taken out of the refrigerator at −80°C, placed at RT for a few minutes and fixed in 1% (w/v) formaldehyde/PBS for 5 min at RT, and then rinsed gently with distilled water three times, incubated in 50% ethanol and 70% ethanol for 5 min each in turn. Next, frozen sections were stained in Sudan Black B solution (0.7 g SBB in 100 mL 70% ethanol) for 5 min at RT, and then rinsed quickly in 75% ethanol to remove excess staining solution, rinsed with distilled water three times. Finally, sections were left to stain in nuclear fast red solution for 3 min, washed in distilled water and mounted with glycerol. Images were taken with Olympus CKX41 microscope imaging system, and Image Pro Plus was used to quantify the Sudan Black B positive area.

For frozen sections, the samples were harvested without fixation and were immediately embedded in SCEM compound (SECTION-LAB). The sections were prepared at 6-μm thickness with a Cryofilm type 2c(9) (SECTION-LAB) using a CM3050S cryomicrotome (Leica) according to the method described by Kawamoto32 (link).
For paraffin-embedded sections, the samples were dissected, fixed in 4% paraformaldehyde, processed and embedded in paraffin. The sections were prepared at 6-μm thickness.
One individual measured the cartilage thickness and cell number of the histological sections in a blinded manner.

Oil Red O staining was performed using a previously published protocol (Huang et al., 2022 ). In short, OCT-embedded and snap-frozen tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome. Frozen sections were taken out of the refrigerator at −80°C, placed at RT for a few minutes and soaked in the PBS solution for 5 min. After filtering Oil Red O staining solution (Sigma-Aldrich) through a 100 μm filter, frozen sections were stained in 60% Oil Red O solution for 8–10 min. To avoid background staining, wash the slides with 70% ethanol for a moment, and then rinsed with running tap water and counterstained with hematoxylin. Images were taken with Olympus CKX41 microscope imaging system, and Image Pro plus was used to quantify the oil red O positive area.

A CM3050 S cryomicrotome (Leica) was used to prepare 16 µm thick sections for imaging with an Alpha300RA Raman instrument (WITec GmbH, Ulm, Germany) as described previously (Gierlinger et al., 2012). The samples were excited using a linear polarized VIS laser (λ_ex = 532 nm, laser power 30 mW) via a 100× oil immersion objective lens (Zeiss, numerical aperture 1.4, coverslip correction 0.17 mm). Raman signals were backscattered through the same objective, directed through a 50 µm optical multifibre to a UHTS 300 spectrometer (WITec) fitted with a 600 g mm⁻¹ grating, and finally to a DU401 BV CCD camera (Andor Technology Ltd, Belfast, UK). control four acquisition software (WITec) was used for data collection with 0.3 µm steps at 0.07 s per spectrum, and the Projectfour plus software (WITec) was used for data analysis. After cosmic ray removal, Raman images were calculated based on band integration, and a hierarchical cluster analysis was performed as described previously (Gierlinger et al., 2012).

Oil Red O staining was performed using a previously published protocol.^{89 (link)} Briefly, OCT-embedded, snap-frozen primate tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome. Frozen sections were stained in freshly prepared Oil Red O staining solution (Sigma-Aldrich) at 60 °C for 8–10 min, washed in running tap water, and counterstained with hematoxylin. Images were taken with a Leica Aperio CS2 system, and the percentages of Oil Red O-positive cells were quantified using ImageJ.