Lsm 710 laser confocal microscope

Transgenic plants expressing 35S:GFP‐fABD2 (Voigt et al., 2005) were crossed with the opal5 mutant, and the F₃ plants homozygous at both loci were used to visualize actin filaments in guard cells of mature leaves from 3‐wk‐old seedlings. Detached leaves were incubated on 10 mM MES/KOH buffer (pH 6.2) for 0.5 h and then floated on the opening buffer (10 mM MES/KOH, pH 6.15, and 50 mM KCl) under white light (125 μmol m⁻² s⁻¹) for 3 h. Half of the light‐treated leaves were incubated under darkness for 30 min. The abaxial epidermis of the leaves was used to observe the guard cell actin cytoskeleton on an LSM 710 laser confocal microscope (Zeiss, Oberkochen, Germany) equipped with a ×63 oil‐immersion objective. Images of guard cells were acquired by serial optical sectioning (Z‐stack) at 1 μm intervals. The samples were excited at 488 nm and emission was detected using a 505 to 530 nm band pass filter. To observe leaf trichomes, the first pairs of true leaves of 10‐d‐old seedlings were fixed overnight at 4°C and dehydrated with ethanol. The specimen was then subjected to a critical point drying process, sputter‐coated and examined on an S‐3000N scanning electron microscope (Hitachi, Tokyo, Japan).

For immunofluorescence microscopy, cells were cultured on coverslips, fixed with 4% paraformaldehyde in PBS for 5 min or with ice-cold methanol for 5 min, and permeabilized with 0.1% Triton X100 in PBS for 5 min. Coverslips were incubated with primary antibodies for 2 h, washed three times with PBS, and incubated with secondary antibodies for 30 min. Samples were mounted using ProLong Gold antifade reagent with or without DAPI (4,6-diamidino-2-phenylindole; Invitrogen) and observed using a Zeiss LSM710 laser confocal microscope. Samples were analysed by Structured Illumination Microscopy using an ELYRA Superresolution microscope from Zeiss. Automatic counting of LC3 vesicles from HeLa cells stably expressing GFP-LC3 was performed using the Cellomics ArrayScan VTI HCS Reader (20 × objective) and the Spot Detector V3 Cellomics BioApplication (Thermo Fisher Scientific). The numbers of vesicles per cell were counted for two thousand cells per coverslip and the mean number of vesicles per cell was calculated by the ArrayScan software.

These sections were blocked with 10% bovine serum in PBS before they were incubated with a mixture of rabbit polyclonal antibody against c-Fos (1:1500) and goat anti-NPSR (1:500, sc-162893, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS containing 1% bovine serum for 72 h at 4°C on an agitator. After several rinses in PBS, the sections were incubated with Alexa Fluor^® 488-conjugated affinipure donkey anti-rabbit IgG (1:400, 711–545–152, Jackson ImmunoResearch Laboratories, Inc., PA, USA) and Cy™ 3-conjugated affinipure donkey anti-goat IgG (1:400, 705–165–147, Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 24 h at 4°C. Primary antibody omission was used as a control. Finally, sections were mounted on slides, covered with a coverslip, using 50% glycerol in PBS, and observed under a fluorescence microscope and photographed under Zeiss LSM 710 laser confocal microscope. The specificity of the anti-NPSR antibody had been demonstrated in previous studies (Laitinen et al., 2004 (link); Shao et al., 2013 (link), 2016 (link)).

Cells were seeded at 1 × 10⁵ cells/well on glass-bottomed culture dishes and then fixed with freshly prepared 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min. Human artery and mouse common carotid artery tissues were harvested and processed in optimal cutting temperature compound and sliced into 5 μm-thick sections. Paraformaldehyde-fixed sections, cryosections, and cells were incubated with anti-NLRC5 (ab105411, Abcam), anti-α-smooth muscle actin (α-SMA, ab7817, Abcam, 1:200), anti-PPARγ (sc-7196, Santa Cruz, 1:100), anti-CD31 (557355, BD Bioscience, USA, 1:100), anti-FLAG (ab49763, Santa Cruz, 1:100), and anti-myc (2276, Cell Signaling Technology, 1:100) overnight at 4 °C. Normal isotype IgG (sc2027, Santa Cruz) was used as negative control. After washing with phosphate-buffered saline (PBS), secondary antibodies (Alexa Fluor 647-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-mouse; Thermo Fisher Scientific, 1:200) were incubated for 1 h at 37 °C in the dark. Nuclei were labeled with DAPI (Vector Laboratories), and cells were visualized using an LSM710 laser confocal microscope (Carl Zeiss, Germany).

Total, cytoplasmic and nuclear proteins were prepared using ReadyPrep Protein Extraction Kit (Bio-Rad, Hercules, CA) and aliquots of them were used for western blot analysis with antibodies to IκBβ, RELA, H3 and β-actin (Abcam, Cambridge, MA). The immunoreactive bands were detected by ECL kit and visualized with ImageQuant LAS 4000. The collected culture medium was used for the assay of ICAM1, VCAM1, SELE, SELP and IL-6 by ELISA kits (Abcam, Inc.). HUVECs were cultured on slides in 6-wells plates and transfected with let-7e mimic and negative control as above mentioned. Then, cell immunofluorescence assay was performed using anti-NF-κB mouse monoclonal primary antibody (Cell Signaling Technology, USA) and Alexa Fluor 594-labelled goat anti-mouse IgG secondary antibody (Jackson, US) as described previously50 . DAPI (6-diamidino-2- phenylindole, Sigma-Aldrich, USA) was used to stain the nuclear. The slides were observed under LSM 710 laser confocal microscope and analyzed using ZEN software (Zeiss, Germany).