Application suite advanced fluorescence las af

The adjacent hippocampal brain sections (10 μm) from the same levels used for the detection of glutamatergic neurons and GABAergic neurons were incubated in 2 N HCl for DNA denaturation (n = 3), neutralised with 0.1 M boric buffer (pH 8.5) and then incubated in bovine serum and the primary antibodies: the mouse anti-glutamate (1:2000 dilutions, cat. no. 22523, Immuno Star Inc., WI, USA) or the mouse anti-GAD67 (1:500 dilutions, cat. no.sc-28376, Santa Cruz Biotechnology Inc., Texas, USA) antibody in combination with the rabbit anti-NeuN antibody (1:500 dilutions, cat. no. ab104225, Abcam Inc., MA, USA) for 120 min at 4 °C overnight. The negative control group was incubated with 0.01 M PBS. Then, sections were washed and incubated in the respective secondary antibody: goat anti-rabbit IgG^® FITC (1:200 dilutions, cat. no. bs-0295G-FITC, Bioss Biotechnology, Beijing, China) or goat anti-mouse IgG^®Cy3 (1:200 dilutions, cat. no. 115-166-003, Jackson Immuno Research Laboratories Inc., Baltimore, USA). The stained sections were examined with a Leica fluorescence microscope (Leica, DM5000B; Leica CTR5000; Leica, Germany). The Leica Application Suite Advanced Fluorescence (LAS AF, Leica Microsystems, Germany) software was used for quantification.

ROS production in guard cells was analysed using a chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Molecular Probes, Invitrogen) as previously described [4 (link)] with slight modifications. Seven-day-old WT and Atcuaoβ mutant seedlings were incubated for 3 h in the assay solution containing 5 mM KCl, 50 μM CaCl₂ and 10 mM MES-Tris (pH 6.15), and then 10 μM CM-H₂DCFDA was added to the sample. Seedlings were incubated for 30 min at room temperature and then the excess dye was washed out twice with the assay solution. After this period, the assay solution was replaced by liquid medium in the absence or presence of treatment performed as follows: 50 μM MeJA, 50 μM MeJA + 100 μM DMTU, 50 μM MeJA + 5 mM 2-BrEtA. Seedlings were incubated for 1 h. Images were acquired by Laser Scanning Confocal Microscopy (LSCM), using a Leica TCS-SP5 equipped with an Argon laser (Excitation/Emission: ~492–495/517–527 nm) and the Leica Application Suite Advanced Fluorescence (LAS-AF; Leica Microsystems, Wetzlar, Germany).

ROS production in guard cells was analyzed using a chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Molecular Probes, Invitrogen) as previously described [25 (link),67 (link)], with slight modifications. Arabidopsis leaves from 12-day-old seedlings from WT, as well as AtCuAOδ insertional mutants and over-expressing lines grown on agar medium, were detached and incubated for 3 h in the assay solution containing 5 mM KCl, 50 μM CaCl₂, and 10 mM MES-Tris (pH 6.15), and then 50 μM CM-H₂DCFDA was added to the sample. Leaves were incubated for 30 min at room temperature and then the excess dye was washed out with the fresh assay solution. Collected tissues were again incubated in the assay solution containing 100 μM ABA for 20 min in dark conditions. Images were captured by Laser Scanning Confocal Microscopy (LSCM), using a Leica TCS-SP5 equipped with an Argon laser (Excitation/Emission: ~492–495/517–527 nm) and the Leica Application Suite Advanced Fluorescence (LAS-AF; Leica Microsystems, Milan, Italy).