Tcs sp8 x confocal microscope

After 24 h stretch, HCAECs were fixed for 10 min with 3.7 % formaldehyde, followed by three times washing with PBS and 30 min blocking with 1 % bovine serum albumin. Cells were then incubated for 1 h at room temperature with an anti-VE-cadherin monoclonal antibody (1:60, eBioscience, San Diego, CA, USA) and unbound antibody was washed away with PBS. Afterwards, a secondary antibody (1:400, Thermofisher) was applied on cells for 30 min. Cells were covered with mounting fluid containing DAPI (Thermofisher). VE-cadherin was imaged using Leica TCS SP8 X Confocal Microscope (Leica microsystems) at 630 × magnification.

Plasmids pcL-cYFP-P3N-PIPO and pcL-nYFP-NbRLK6 were separately transformed into A. tumefaciens strain GV3101. The two agrobacterium cultures were mixed at a 1:1 (v/v) ratio and infiltrated into the leaves of N. benthamiana plants. Mixed agrobacterium cultures carrying pcL-cYFP-P3N-PIPO and pcL-nYFP-GUS served as a non-interacting control. At 48 h post-infiltration (hpi), the leaves were collected and examined under a Leica TCS SP8X confocal microscope (Leica Microsystems, Bannockburn, IL, USA).

For in planta subcellular localization analysis in N. benthamiana, cut leaf patches were mounted in water and analyzed on a Leica DM6000B/TCS SP5 confocal microscope (Leica Microsystems) with the following excitation wavelengths: GFP, 488 nm; RFP, 561 nm. In the case of Arabidopsis transformants containing Est-GFP, Est-103, and Est-103^Hind2, 3-week-old transgenic lines 24 h after spray treatment with estradiol were DAPI-stained using CySain^® UV Precise P (Sysmex) by vacuum infiltration. After incubation in dark for 1 h, cut leaf patches were mounted in water and analyzed on a Leica TCS SP8 X confocal microscope (Leica Microsystems) with the following excitation wavelengths: GFP, 488 nm; DAPI, 405 nm.

Cells were seeded as described in the cell death assay except on Nunc™ Lab-Tek™ II 8-well Chambered Coverglass (155409PK, Thermo Scientific™) or 35 mm coverglass culture dishes (MatTek Life Sciences, P35G-1.5-14-C), irradiated ~24 h after seeding and placed on the Leica TCS SP8 X confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) beginning 24, 48, 72, or 96 h after IR and imaged every 20–30 min using a ×63/1.4 N.A. oil objective for ~16 h at a time at 37 °C in 5% CO2 using an OKO-labs cage incubator. Prior to imaging, cells were stained with LysoTracker™ Deep Red (Invitrogen™, Life Technologies Corp., Carlsbad, CA) for 15–30 min, washed twice with PBS and stained with propidium iodide (1:500) in FluoroBrite DMEM media (Invitrogen, Life Technologies Corp., Carlsbad, CA) with 10% FBS and 4mM L-glutamine. Images were collected using LASX software (Leica Microsystems, Buffalo Grove, IL) and visualized and analyzed using Volocity software (v6.3.5, Quorum). Image contrast and brightness were adjusted for presented images in Microsoft PowerPoint with similar adjustments for all images in the experiment (acquired with identical microscope settings). As acquired images available for review.

CD15⁺ cells were enriched from PBMCs samples by immunomagnetic separation using MojoSort Streptavidin Nanobeads (BioLegend) after incubating with a biotin-conjugated anti-human CD15 antibody (BioLegend) following the protocol provided by the manufacturer. Enrichment yield after sorting was confirmed by flow cytometry. Then, enriched CD15⁺ fractions were seeded in poly-L-lysine coverslips and incubated at 37 °C, 5% CO₂ for 30 minutes. Cells were washed twice with PBS and incubated with different antibodies [CD16 APC (Immunostep), CD15-biotin (BioLegend) and CD14 FITC (Immunostep) or CD10 FITC (BioLegend)] for 1 hour at 4 °C. Parallel incubations were performed with paired isotypes for each staining. Next, coverslips were washed twice with PBS and cells were incubated with streptavidin-conjugated PE (Immunostep) for 30 minutes at 4 °C. Coverslips were washed twice and cells were fixed with a 4% paraformaldehyde solution for 5 minutes at 4 °C. Finally, coverslips were mounted with Fluoroshield Mounting Medium With DAPI (abcam) and immediately analyzed using a Leica TCS-SP8X Confocal Microscope (Leica Microsystems) using the 63X objective. Images were acquired with LAS X software (Leica), which was also used to produce image overlays. To evaluate nuclear morphology, z-stacks were obtained from 4 μm sections and maximum projections were derived.

The 40-μm muscle sections were analyzed for the positivity for fluorophores Cy3 (550–570 nm), Cy5 (649/665 nm), and alpha-bungarotoxin-Alexa 488 (488–520 nm) using a Leica TCS SP8 X confocal microscope (Leica Microsystems B.V., Rijswijk, The Netherlands). For each view, Z-stacks (objective ×40/1.30 oil; 290 μm × 290 μm) of 40-μm thick muscle tissue were made. The images were analyzed using Leica LCS software (Leica).