Cryostar nx50 cryostat

Banked human tissue samples including 28 human glioblastomas (GBM, astrocytoma WHO grade 4), 19 lower-grade astrocytoma (AST, 10 WHO grade 1, and 9 WHO grade 2) and 29 non-cancerous brain cortex (NL) specimens were obtained from the University of Alabama Brain SPORE via the Cooperative Human Tissue Network and Baylor St. Luke’s Medical Center at the Baylor College of Medicine under approved institutional review board protocols. Isocitrate dehydrogenase 1 (IDH1) mutation statuses as determined by immunohistochemical staining were provided by the tissue source for a subset of tumor specimens. A list of patient sample demographics can be found in Table S1. Samples were stored at −80 °C until sectioning. Tissues were sectioned at 16 um thickness using a CryoStar NX50 cryostat (Thermo Scientific, San Jose, CA, USA) and mounted onto glass slides. Mounted sections were stored at −80 °C until MS imaging. Prior to imaging experiments, slides were dried for ~10 min. Due to the size of the tumor samples obtained and differences in tissue mass requirements for subsequent analyses, we restricted immunofluorescence staining and CL structural characterization analyses to only a single grade of lower-grade AST tumors.

After isolation of the DRG and spinal cord the tissue was frozen at –50 °C using isopentane cooled by liquid nitrogen in a small amount of freezing medium (14020108926, Leica, Wetzlar, Germany). The 12-μm sections were sliced with a cryostat (CryoStar NX50 Cryostat, Thermo Fisher Scientific, Waltham, MA, USA), mounted on Superfrost Plus slides (J1800AMNZ, Thermo Fisher Scientific, Waltham, MA, USA) and stored at –80 °C.

OCT-embedded skin tissues of DMSO vehicle control or Ver155008-treated mice were cryosectioned at a thickness of 10 μm using the CryoStar NX50 cryostat (Thermo Fisher Scientific). Cryosections were incubated at 55°C for 10 min, washed in phosphate-buffered saline (PBS) before performing a standardized H&E staining protocol using Gill’s 3 formulation Hematoxylin (Thermo Fisher Scientific) and Eosin Y at pH 4.7 (Thermo Fisher Scientific) for counterstaining and clearing of the stain with xylene-based solutions, and mounted with a toluene-based mounting medium (Permount, Fisher Scientific). Images were acquired with the 10× objective on an AmScope bright-field microscope with a MU500B digital camera.

Xenografted tumors were harvested, washed with PBS and incubated overnight in 4% PFA at 4 °C. The tumors were subsequently washed three times with PBS (5 min) and incubated in 10% sucrose (2 h), 20% sucrose (2 h) and 30% sucrose (overnight) at 4 °C. Next, the tumors were placed in liquid optimal-cutting-temperature medium Tissuetek (Sakura, AJ Alphen aan den Rijn, the Netherlands) and flash-frozen for storage at −20 °C. Frozen tumor tissue sections were cut (10 µm thick) with a CryoStar™ NX50 Cryostat (Thermo Scientific, Waltham, MA, USA). Hematoxylin and eosin staining was performed for morphological confirmation following routine laboratory protocol. For antibody staining, the tumor tissue sections were blocked with human IgG (R & D Systems) for 15 min at room temperature. Afterwards, 10 µL of monoclonal anti-HER2 antibody (R & D Systems) was added and incubated for 30 min at 37 °C. Hoechst staining was performed by incubating the tumor tissue sections with 0.1 mg/mL Hoechst final concentration (Sigma Aldrich) for 10 min at room temperature. For aptamer staining, the tumor tissue sections were washed with PBS and incubated with the aptamer at a final concentration of 250 nM in selection buffer. After incubation (30 min at 37 °C), the tumor tissue sections were washed with PBS to remove unbound aptamers. Images were acquired as described above.

Each tissue block was pierced 3 times with an acupuncture needle (38-gauge, 0.20 mm thickness) for orientation to match consecutive sections. All samples were sectioned at 20 μm using a CryoStar NX50 cryostat (ThermoFisher Scientific, Waltham, MA, USA) using C.L. Sturkey ‘Diamond’ PTFE-coated stainless-steel disposable microtome blades (ProSciTech, Australia). The cryostat chamber was set at −20 °C and the sample holder at −12 °C. The first section was used for immunohistochemistry (IHC) and was mounted on a Superfrost™ microscope slide (ThermoFisher Scientific, USA). The adjacent section was reversed and mounted on a Si₃N₄ window (5 × 5 mm, 200 nm film thickness, 200 μm frame thickness; Australian National Fabrication Facility, QLD, Australia) with the matching tissue face exposed for analysis.

The anatomy of lotus leaves and petioles was observed with a light microscope (Leica DMi8, USA) and a transmission electron microscope (TEM; Hitachi HT7800, Japan). Lotus leaf and petiole samples at developmental stage 5 (S5) were taken from plants grown under routine conditions and after 48 h of complete submergence. For frozen tissue specimens, fresh samples were embedded with Tissue-Tek (Sakura Finetek, CA, USA), immediately frozen at −20°C, and sliced into 20-μm sections with a Cryostar NX50 cryostat (Thermo, CA, USA). For safranin O/fast green staining, fresh samples were fixed in FAA solution for 2 days, dehydrated with ethanol, and embedded in paraffin. Sections of 10 μm thickness were cut, stained with 1% safranin O for 10 min, and then counterstained with 0.1% fast green solution for 5 min. For TEM observation, samples of approximately 1 mm [3 ] were fixed with 1% OsO₄ in 0.1 M phosphate buffer (pH 7.4) for 7 h at room temperature, dehydrated with ethanol, embedded in EMBed 812 (SPI Chem, PA, USA), sliced into 60–80-nm sections, and stained with a 2% uranium acetate saturated alcohol solution in the dark for 8 min.