Canoscan 9000f mark 2

The array membrane was placed inside a XX30001200 Swinny Filter Holder 13 mm (Merck Millipore). All IgG-modified nanoparticles were redispersed in an assay buffer that consisted of 29 mM sucrose, 0.44 mM BSA, 0.45 M NaCl, 0.5% w/v Tween 20 in 0.1 M phosphate pH 7.4 buffer. IgG-modified AuNPs were prepared as to have 8 pM, IgG-modified AgNPs were prepared as to have 17 pM, IgG-modified SiNPs were prepared as to have 0.1 mg·mL⁻¹ and IgG-modified IONPs were prepared as to have 0.1 mg·mL⁻¹. The flow was controlled using a PhD2000 ultrasyringe pump (Harvard Apparatus) and set to a rate of 1 mL·min⁻¹. The arrays were left to dry at room temperature for 5 minutes before being digitalized in a flatbed scanner CanoScan 9000 F Mark II (Canon) in 24-bit colour.

Ultrathin sections were examined and photographed with a transmission electron microscope (EM 109 and EM10C, Zeiss, Oberkochen, Germany). Photomicrographs (Agfa Scientia EM film) from selected sections were scanned at 2400 dpi (CanoScan 9000F MarkII, Canon, Tokyo, Japan) and processed further with Adobe Photoshop CS3 and CorelDRAW X3.
GABA-labeled immunogold sections were evaluated statistically. The mean labeling density of gold particles (GPs) over selected profiles was determined in 4–8 consecutive ultrathin sections and compared against GP levels in adjacent, presumably unlabeled profiles of similar size and organelle composition (Watson, 1988 (link); Watson et al., 2000 (link); Träger et al., 2007 (link)). Profiles were accepted as GABA-immunoreactive, if they had a significantly higher GP concentration than an adjacent reference profile (t-test, p < 0.05); when tested against two adjacent profiles, one-way ANOVA with Tukey-HSD multiple range test was performed (p < 0.05).

A 1000 ppm stock solution of free chlorine was prepared by dissolving 92.5 mg dichloroisocyanuric acid sodium salt dihydrate (GR for analysis, Merck KGaA) in 50 ml Milli-Q water on the day of analysis. The stock solution was diluted to 5 and 2 ppm. Solutions with lower concentrations were prepared by diluting the 5 ppm solution. All used glass vials and flasks were pre-rinsed with the respective solution. The flow tests were run in 0.5 ml of the respective solution in a 3 ml vial (Fig. 2b), whereby the level of the sample solution is below the SA zones on the test. After removal of the flow tests from the solution, they were immediately scanned with the scanner CanoScan 9000 F MarkII (Canon, 600 dpi without any image correction). The intensity of the purple colour of the oxidized SA was determined by ImageJ 1.94 v using the colour deconvolution plugin^{15 (link)}. The RGB stain vector was determined to [0.38 0.89 0.24] on paper and [0.37 0.86 0.35] for PET and GF and the RGB intensity value determined from the deconvoluted “purple” image, on which only this colour remained in order to avoid interference from grey background due to water stains on the substrate.

Between 12 and 14 leaves were collected from six shoots per sample from plants in Madera, California, USA in June 2022. The sampled shoots grew from retained nodes. Leaves were pressed in an herbarium press at Madera, California, USA and shipped in the press to East Lansing, Michigan, USA for scanning and analysis. The leaves were scanned using a CanoScan 9000 F Mark II (Canon U.S.A., Inc) at 600 DPI. The leaves were landmarked manually by placing 21 landmarks from Bryson et al. [11 (link)] on leaf scans using ImageJ v1.53k [12 ]. Scans were saved as x- and y-coordinates in centimeters. The shoelace algorithm, originally described by Meister [13 ], was used to calculate leaf, vein, and blade areas using the landmarks. The landmarks were used as the vertices of polygons and the following formula, as described in Chitwood et al. [14 (link)], was used to then calculate the areas (where n represents the number of polygon vertices defined by the landmarked x and y coordinates):
To investigate changes in leaf shape between WT and WB leaves, a generalized Procrustes analysis and a principal components analysis (PCA) was performed using the shapes package v1.2.7 [15 ] in R v4.2.2 [16 ] and RStudio v2022.12.0.353 [17 ], with scaling and rotation. The shapes package v1.2.7 [15 ] in R and RStudio was also used to test for mean shape differences using a Hotelling’s T² test.

A colony formation assay was used to measure the long-term effect of treatments on cell proliferation. Briefly, cells were transfected with the respective miR-506-3p mimic or control oligos (20 nM) in 6-well plates and cultured overnight. On the second day, cells were trypsinized, and 5000 cells containing DMEM/F-12 cell culture medium supplemented with 10% FBS and 1% P/S were seeded into 100 mm dishes in three replicates. The cells were then cultured for 10–14 days or until colonies were visible. For staining the colonies, the cells were rinsed with 1X PBS and fixed/stained with 0.05% w/v crystal violet (containing 10% methanol and 1% PFA) for 20 min at room temperature. After the fixing and staining solution was decanted, colonies were washed with cold water and left overnight to air dry. Plates were scanned using a CanoScan 9000F Mark II (Canon, Tokyo, Japan). Colony numbers and sizes were quantified using ImageJ software (bundled with Java 8).