Lmd system

Early Arabidopsis embryo isolation and the separation of apical and BC lineages of early proembryos were performed according to our previous protocol^{11 (link)}. For embryo isolation, Arabidopsis seeds were collected in the enzyme solution (0.1% cellulase R10, 0.08% macerozyme R10, 80 mM d-Sorbitol, 10% glycerol, 0.058% MES, pH = 5.8) for 30 min at 25 °C, followed by three washes in the washing solution (80 mM d-Sorbitol, 10% glycerol, 0.058% MES, pH = 5.8). Early embryos were then isolated manually from the seeds with two fine glass needles under an inverted microscope. Apical and BC lineages of early proembryos were separated using LMD System (Leica, Germany), and collected under an inverted microscope by the micromanipulation. Isolated apical and BC lineages were extensively washed four times and transferred into lysis buffer (Life Technologies, USA) and stored in liquid nitrogen for mRNA isolation.

The dried medicinal materials were firstly softened by infiltrating with water-soaked paper. The softened Cinnamomi Cortex was cut into small sections, fixed by cryogen, and then frozen on a − 20 °C cryobar. Serial slices of 40 μm in thickness were cut at − 10 °C. Each cross-section of tissue was mounted directly to a non-fluorescent polyethylene terephthalate. The slide was exposed under a Leica LMD 7000 microscopic system. Microdissection was conducted by a DPSS laser beam at 349 nm wavelength, aperture of 30, speed of 3, power of 50 μJ and pulse frequency of 1695 Hz under a Leica LMD system at 6.3 × magnification. Four different target tissues, approximately 1 × 10⁶ μm² per each, were individually separated. The microdissected tissues fell into caps of 500 μL micro centrifuge tubes by gravity. Lastly, the separated tissue part in each cap was transferred to the bottom of the tube by centrifuging for 10 min (12,000 rpm, 17 °C). 100 μL methanol was added into each micro centrifuge tube. The tube was sonicated for 60 min and then centrifuged again for 10 min (12,000 rpm, 17 °C). 90 μL of the supernatant was transferred into a glass insert with plastic bottom spring in a 1.5 mL brown HPLC grade vial and stored at 4 °C before analysis.

LMD was performed at the Histopathology Core of UT Southwestern Medical Center as previously described¹¹⁷ . Briefly, 10 µm cryo-sections of tumors were mounted onto PEN membrane glass slides (Thermo Fisher Scientific, LCM05220). Sections were then stained using Histogene™ Staining Solution (Thermo Fisher Scientific, KIT0415), washed with high-performance liquid chromatography (HPLC)-grade water and subjected to LMD using a Leica LMD System. LMD sections were recovered in 0.2 mL tubes, immediately snap-frozen and stored at −80 °C until the analysis. Four replicates per sample were prepared.