Cm1850 uv cryostat

A total of 12 patients were selected based on the histological phenotype (papillary, acinar and lepidic growth pattern) and included matched normal lung tissue of the same patient. Frozen samples were cut in 5-10 mm slices using the Leica CM 1850 UV cryostat, and 30 to 100 mg of tissue was collected. Subsequently, the slices were lysed with TRIzol (Life Technologies) and 1.4 mm ceramic spheres (Lysing Matrix D, MP Biomedicals, DBA, Milan, Italy) using the FastPrep-24 homogenizer (MP Biomedicals) (2 cycles, 30’’ on at room temperature and 5’ off on ice). RNA was extracted with chloroform and isopropanol using standard protocols. RNA integrity was checked by gel electrophoresis. Two mg of purified RNA was converted into cDNA, and 25 ng was used for RT-qPCR experiments as described above; expression calculations were generated as ΔCt values normalized to reference genes ATCB and YWHAZ. Primers are the same used for the experimental procedures as explained in the previous paragraph.

For immunohistochemistry, cleaned reproductive tracts (see above) were fixed, in toto, in 4% methanol-free paraformaldehyde (Polysciences, Warrington, PA) in mHTF-Hepes overnight at 37 °C. After fixation, reproductive tracts were washed 3x in sterile PBS, air dried at -20 °C for 2–4 hrs, and embedded in Optimal Cutting Temperature compound (Tissue-Tek OCT; VWR, Bridgeport, NJ). Embedded reproductive tracts were stored at -80 °C. For processing, 7-μm sections were cut on a cryostat at −20 °C (CM1850 UV cryostat; Leica, Buffalo Grove, IL), collected on clean glass slides (Diamond White Glass 25 × 75 × 1 mm, (+)- charged; MidSci, Valley Park, MO), and stored at −80 °C until staining. To detect direct GFP expression, slides were brought to room temperature for 5 minutes and stained for 10 min with 10-μg/ml Hoechst 33342. Sections were sealed in antifade solution using a clean coverslip before imaging. In some instances, GFP CETN2 sections were first immunostained using antibodies to Ak15 γ-tubulin (1:500; Sigma-Aldrich) and microtubules (YOL1/34; 1:200; EMD Millipore) as describe above.

P4 neonatal and adult excised ovaries were fixed in toto in 4% pFA (Electron Microscopy Services) in mHTF-Hepes overnight at 37 °C, rinsed 3x in sterile PBS, air dried at −20 °C for 2–4 hr, and then embedded in optimal cutting temperature compound (OTC; Tissue-Tek; VWR, Bridgeport, NJ) prior to storage at −80 °C. 7-µm sections were cut on a cryostat at −20 °C (CM1850 UV cryostat; Leica, Buffalo Grove, IL), floated onto clean glass slides (MidSci, Valley Park, MO), and stored at −80 °C. For immunostaining, slides were warmed at room temperature for 5 min, treated for 10 secs with Surgipath O-Fix (Thermo Fisher), and washed briefly in distilled H₂O. Immunostaining was performed as described above.

For microdissection of the SN and the VTA neurons, P2 chicks reared in complete darkness were euthanized via carbon dioxide gaseous asphyxiation, their brain extracted and then fast-frozen in dry-ice-cold isopentane solution. 100 μm coronal sections were cut using a Leica CM1850 UV Cryostat at −15°C, and stored at −20°C. To better localize the targeted areas, sections were stained for 15 min with a 0.01% cresyl violet solution dissolved in 100% ethanol, and then progressively dehydrated in 75, 90, and 100% ethanol (1 min/each). All solutions were prepared fresh and filter-sterilized to avoid RNases contaminations. Substantia nigra and VTA regions were finally dissected out using a 20G needle and immediately processed for total RNA extraction. For dissections of the septum, P2 chicks reared in darkness were euthanized by carbon dioxide gaseous asphyxiation, the brain extracted, and the area of interest directly dissected. Briefly, two coronal cuts were performed approximately 2 and 4 mm anterior to the bregma to isolate the septum in the anterior-posterior axis according to Puelles et al. (2007) and the septum was carefully removed with forceps, fresh frozen in dry ice and immediately processed for total RNA extraction.

Animals were deeply anesthetized with ketamine/xylazine (10% Ketamine hydrochloride, WDT; 2% Rompun, Provet AG) i.p. before being transcardially perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA). The brains were removed from the skull, post-fixed in 4% PFA at 4 °C overnight, dehydrated with 30% sucrose solution at 4 °C for 48 h, and frozen at − 72 °C in 2-methylbutane (Sigma-Aldrich, Steinheim, Germany). Afterward, the brains were coronally sliced into 40-μm-thick sections using a Leica CM1850 UV cryostat and stored in cryoprotectant solution at 4 °C until histological analysis was performed.
For collecting blood samples and fresh brain tissue for molecular analysis, the animals were as well deeply anesthetized with ketamine/xylazine. Then, the abdomen was opened, and the blood samples were taken from the inferior vena cava. Aprotinin (Sigma-Aldrich, St. Louis, USA; 1 μl/1 ml blood sample) was added to the samples to prevent protein degradation. Samples were spun with an acceleration of 8000×g at 4 °C for 15 min, and sera were collected. After taking blood samples, the animals were transcardially perfused with PBS. Afterward, the brains were quickly removed from the skull and rapidly frozen on dry ice. The brains and serum samples were stored at − 80 °C until further analysis.