Membrane filters

Freshly isolated retinal slices (1 mm thickness) were prepared and placed with the photoreceptor side onto membrane filters (0.45 μm, diameter 50 mm; GE Healthcare Life Sciences, Freiburg, Germany). Volume changes of Müller cell somata evoked under isotonic conditions and after hypoosmotic challenge (60% of control osmolarity) were measured in the inner nuclear layer of retinal slices as previously described [77 (link)]. The retinal slices from wildtype and KO mice were placed in a perfusion chamber (custom made) and loaded with the vital dye Mitotracker Orange (10 μM, excitation 543 nm, emission 560 nm-long pass filter; Thermo Fisher Scientific). The Mitotracker Orange dye selectively stained Müller glia [78 (link)], and the stock solution was prepared in DMSO and diluted 1:1000 in PBS. The slices were examined during exposure to a hypotonic solution for 4 min with or without test agents using confocal microscopy (custom-made VisiScope CSU-X1 confocal system equipped with a high-resolution sCMOS camera; Visitron Systems). The cell soma area of labeled Müller cells was cross-sectionally measured (ImageJ). The blocking agent was pre-incubated for 10 min in extracellular solution; AC710 (PDGFR family inhibitor; Tocris Bioscience, Bristol, UK) 100 nM. PDGF-BB, a PDGF receptor α/β agonist (R&D Systems), was applied simultaneously with the hypotonic solution.

Almond and pistachio nutshells were obtained from a local tree-nut farm (Valley Orchard, LLC, Fresno, CA, USA). Four to five tree nutshell particles, depending on their sizes, were aseptically placed onto potato dextrose agar (PDA; BD Life Sciences, Franklin Lakes, NJ, USA) and maintained at 28 °C. The level of fungal contamination was monitored for 72 h.
Then, the level of the antifungal activity of SA against fungi naturally contaminated on the surface of tree nutshells (almond, pistachios) was examined on PDA according to the modified method described previously [26 (link)]. SA (0.8 or 1.6 M) dissolved in 60% ethanol (v/v) was applied to membrane filters (2.5 cm diameter; GE Healthcare, Chicago, IL, USA). SA- or ethanol (control) -saturated filters were positioned onto a PDA Petri plate (100 mm × 15 mm; Corning Inc. Life Sciences, Tewksbury, MA, USA). Two to three tree-nutshell particles, depending on their sizes, were aseptically positioned onto the other half of each PDA. The test plates were incubated at 28 °C to determine the susceptibility of the fungal contaminants to the SA. The control plates contained an ethanol (60%, v/v) filter only. Fungal growth was monitored for 96 h.