Leica sp2 aobs confocal microscope

In vivo localization of NO in Arabidopsis roots was detected using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate DAF-FM DA probes as described by Zhu et al. [36 (link)]. To analyze the levels of NO, the Arabidopsis roots were loaded with 2.0 μM DAF-FM DA in the dark for 20 min, and then washed at least three times in phosphate-buffered saline (PBS) (pH 7.2) solution. NO-associated fluorescence was detected using a Leica SP2-AOBS confocal microscope (Leica, Wetzlar, Germany) with an excitation filter of 490 nm and an emission filter of 520 nm.

Cells were seeded in circular cover glasses, preincubated with 0.005% poly-L-lysine (Sigma-Aldrich) for 30 min at 37°C, and rinsed several times. Cells were then fixed with 4% p-formaldehyde in PBS for 5 min and, after rinsing, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS (both steps at room temperature, 20–22°C). Nonspecific binding sites were blocked by incubation for 1 h in PBS containing 10 μg/ml human IgG and a commercial blocking reagent (Roche, Basel, Switzerland). Incubation with primary TCRβ antibody (JOVI-1) or secondary anti-mouse antibody AF594 (Invitrogen) was done for 1 h or 30 min at 20–22°C, respectively. Appropriate isotype-matched IgG or normal serum was used in parallel as the control. Stained cells were mounted in Vectashield^® -DAPI solution (Vector Laboratories, Burlingame, CA, United States) and visualized under a Leica SP-2 AOBS confocal microscope (HCX PL APO × 63 1.4 oil-immersion objective; 1.400,000, numerical aperture) and TCSNTV software (Leica Microsystems, Wetzlar, Germany). Images were software processed with ImageJ.

Cells cultured on glass cover slips (Thermo Fisher) were washed with PBS and fixed with methanol/acetone (1∶1) for 20 min at −20°C. Cells were then washed with TBS twice and blocked with 2.5% bovine serum albumin (BSA) (Sigma) in TBS for 1 h at room temperature followed by incubation with primary antibodies diluted in blocking buffer overnight at 4°C. Cells were then washed with TBS five times (5 min/time) at room temperature. Secondary antibodies diluted in blocking buffer were then added into the samples and incubated for 1 h at room temperature. Samples were then washed with TBS five times at room temperature. The cover slips were stained with DAPI (DAKO) and mounted onto microscope glass slides (Thermo Fisher) with nail oil. Images were captured using a Leica AOBS SP2 confocal microscope (Leica, Allendale, NJ) and analyzed by using Volocity software. Details on the antibodies used are listed in the Supporting Information.

Cells cultured on glass cover slips (Thermo Fisher) were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with methanol/acetone (1:1) for 20 min at −20°C. Cells were then washed with TBS (25 mM Tris, 0.15M NaCl, pH 7.2 to 7.5) twice and blocked with 2.5% BSA in TBS for 1 h at room temperature followed by incubation overnight at 4°C with polyclonal rabbit anti-NFAT5 antibody (Santa Cruz) diluted in blocking buffer. Cells were then washed with TBS five times at room temperature. Slides were stained with goat anti-rabbit IgG (H + L) labeled with Alexa Fluor 488 and then incubated for 1 h at room temperature. After a final wash, the slides were stained with DAPI (DAKO) and mounted with nail polish. Images were captured using a Leica AOBS SP2 confocal microscope (Leica, Allendale, NJ) as described previously [33 (link)].