Perfection v500

Sections were desiccated at room temperature for 15 min before the spray coating of a norharmane matrix solution for dual polarity analysis of lipids^{72 (link)}. Prior to matrix coating, the slide was scanned on a flatbed scanner (Epson Perfection V500, Nagano, Japan). The matrix solutions were prepared by dissolving the norharmane matrix powder in 80% MeOH (7.5 mg/ml) solution in a glass vial, followed by brief sonication. An automated pneumatic sprayer (TM-Sprayer, HTX-Technologies LLC, Chapel Hill, NC, USA) was used, and combined with a pump (AKTA FPLC P-905, Amersham Pharmacia Biotech, Uppsala, Sweden) to spray the heated matrix solution over the tissue sections. The pump was kept running at 70 μL/min using a 50% ACN pushing solvent with isocratic pressure before the experiments to ensure a stable flow of the solvent. The matrix solution was sprayed using the following instrumental parameters: solvent flow rate of 70 μL/min; nitrogen pressure of 6 psi; nozzle spray temperature of 60 °C; 15 passes (all horizontal); nozzle head velocity of 1200 mm/min; and track spacing of 2.0 mm.

GST-ScPpz1, GST-ScPpz1-Cter, GST-UmPpz1, UmPpz1-s, GST-ScHal3, GST-UmHal3, GST-UmHal3_PD and GST-UmHal3_PD_ScCter were expressed using BL21 (DE3) RIL E. coli cells and purified as reported [58 (link)] with specific modifications [24 (link)]. The GST-tag was removed by PreScission protease (GE Healthcare), as described. The recombinant proteins were analyzed by SDS-PAGE followed by Coomassie Blue-staining and gels were scanned with an Epson Perfection V500 Photo scanner apparatus. Estimation of the amount of full length proteins was done with Gel Analyze 2010a software (http://www.gelanalyzer.com/) in comparison with bovine serum albumin (BSA) standards.

Fresh frozen mouse brains were cut at a thickness of 12 μm using a cryostat microtome (Leica CM, Leica Microsystems). Tissue sections were thaw-mounted onto conductive indium tin oxide (ITO) glass slides (Bruker Daltonics) and stored at −80 °C. Sections were desiccated at room temperature for 15 min before spray coating of norharmane matrix solution. Prior to matrix coating, the slide was scanned on a flatbed scanner (Epson perfection V500). The matrix solutions were prepared by dissolving the norharmane matrix powder in 80% MeOH (7.5 mg/ml) solution in a glass vial and sonicated briefly. An automated pneumatic sprayer (HTX-Technologies LLC, Chapel Hill, NC, USA) was used, which was combined with a pump (AKTA FPLC P-905 pump, Cytiva, Uppsala, Sweden) to spray heated matrix solution over the tissue sections. The pump was kept running at 100 μL/min using a 50% acetonitrile pushing solvent before the experiments to ensure a stable flow of the solvent with isocratic pressure. The matrix solution was sprayed using instrumental parameters of a solvent flow rate of 70 μL/min at isocratic pressure, a nitrogen flow of 6 psi, spray temperature of 60 °C, 15 passes with offsets and rotations, a nozzle head velocity of 1200 mm/min, and track spacing of 2.0 mm.

To determine the linear dimensions of 4- and 6-week-old seedlings, they were laid out individually on the glass of an Epson Perfection V500 Photo flatbed scanner (Epson, Japan) and scanned at a resolution of 800 dpi. MapInfo Professional v. 9.5 software (Pitney Bowes Software, Stamford, CT, USA) was used to measure the lengths of the seedling organs (primary root, hypocotyl, cotyledons, needles) and the distance from the tip of the main root to the first lateral root to an accuracy of 0.01 mm and to count the number of first-order lateral roots and needles of the seedlings [17 (link)].

gelatin zymography was performed as previously described^{47 (link)}. Briefly, gels (SDS-PAGE, 10%) were co-polymerized with gelatin (1 mg/mL) (Sigma-Aldrich). Equal amounts of samples were loaded onto the wells of the gel. After electrophoresis, gels were washed in renaturing buffer (2.5% Triton X-100 in 50 mM Tris-HCl, pH 7.5) for 3 h at room temperature and followed by 18 h of incubation at 37 °C in developing buffer (10 mM CaCl₂, 200 mM NaCl in 50 mM Tris-HCl, pH 7.5). Gels were then stained with Coomassie blue and de-stained with 30% methanol and 10% acetic acid. Gels were photographed by EPSON Perfection V500 (Long Beach, CA, USA).