Dmil led inverted fluorescence microscope

Scanning electron microscopy observations were conducted on an FESEM (SU-8010, Japan). Transmission electron microscopy images were obtained on a JEOL electron microscope (JEM-1400, acceleration voltage 120 kV). UV-Vis-NIR absorbance of the materials were recorded on a Cary5000 spectrometer and fluorescence spectroscopy was performed on an F-4700 spectrofluorometer (Horiba). Thermogravimetric analysis of the lyophilized materials was performed on a Q50 TGA analyzer. The temperature elevation was conducted from room temperature to 700 °C at a scanning speed of 10 °C/min with a N₂ flow of 40 mL/min. Hydrodynamic diameter and zeta potential analysis of the materials was performed by a Malvern nanosizer (NANO-ZS). The nanomaterials at a concentration of 0.2 mg/mL in a neutral buffer was prepared for dynamic laser scanning analysis. Nuclear Magnetic Resonance (NMR) spectroscopy was conducted on an Avanc III 600 MHz Digital NMR Spectrometer (Solvent: DMSO-d6). Molecular weight determination of the materials was performed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF, ultraflextreme, Bruker). Cell and tissue observation was conducted on Carl Zeiss LSM880 laser scanning confocal microscopy and Leica DMIL LED inverted fluorescence microscope. Figures 2i, 3a, 4a, and 4e are created with BioRender.com.

H357 cells were seeded in 6 well plates and cultured as mentioned above. The cells were washed with Phosphate buffer saline (PBS) followed by addition of 100 μl of AO and EB (100 μg/ml, Sigma) and incubated for 15min in CO₂ incubator. Following incubation the medium was aspirated, washed thrice with PBS. The intensity of fluorescent staining was observed and the images were captured with the help of fluorescent microscope (Leica DM IL LED inverted fluorescence microscope) using appropriate color filters.

Samples were deparaffinized through three changes of xylene, and rehydrated in decreasing ethanol concentrations. After quenching in 0.1 mol/L glycin/PBS, antigen retrieval was first performed using citrate buffer solution (pH = 6.0), preheated at 65°C, and then by trypsin. Tissue sections were washed in 0.1% Triton X‐100/TBS, then blocked using BSA 3%/PBS. They were incubated overnight at 4°C with the primary antibody diluted in blocking solution, CD31 (M‐20, Santa Cruz Biotech; 1:100). The next day, they were treated for 1 h with the secondary antibody diluted in blocking solution: donkey anti‐goat IgG (DyLight 549, Abcam; 1:1000) for CD31. DAPI was used for nuclear DNA labeling. Immunostained slides were imaged by LED Inverted fluorescence microscope (DMIL LED Inverted Fluorescence Microscope, Leica, Germany) and images were analyzed using Image J 1.36b (http://rsbweb.nih.gov/ij/).

Protein-protein interactions were detected using the Duolink II in-situ PLA Detection Kit (Sigma Aldrich, USA). Briefly, the hTERT-HM^A/B were cultured on 16 well chamber slides (Lab Tek, Fisher, CA), induced for PRA or PRB expression, serum starved for 24 h in serum free medium and then stimulated with Progesterone (P4, 100 nM) for 2 h. Cells were then washed with cold PBS and fixed with cold methanol: acetone (1:1) for 3 min. After washing, cells were permeabilized with 0.2% Triton X-100 for 5 min, blocked with the blocking solution (provided in the Duolink kit) for an hour and incubated with primary antibodies for overnight at 4 °C. Hybridization with PLA probes (plus and minus), ligation and amplification of conjugants was performed as per manufacturer’s instructions. Slides were mounted with Dapi containing antifade mounting medium (Sigma Aldrich, USA) and pictures were taken at constant exposure and 200X magnification by Leica DM IL LED-Inverted fluorescence microscope with micropublisher 5.0 RTV Q imaging system.

Proximity ligation assay was performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, MO, USA). Briefly, cells were cultured on chamber slides, fixed with paraformaldehyde for 3 min. After washing, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked with PLA blocking buffer for 1 h and incubated with primary antibodies for overnight at 4 °C. Following three washes with PBS the cells were incubated with anti-rabbit-PLUS (Sigma-Aldrich; DUO92002 for UCK2 antibody) and anti-mouse-MINUS (Sigma-Aldrich; DUO92004 for EGFR antibody) PLA probes and subjected to ligation and amplification reaction using Duolink In Situ Detection Reagents Red (Sigma-Aldrich; DUO92008) according to the manufacturer’s protocol. The cells were mounted with DAPI and visualized with Leica DM IL LED-Inverted fluorescence microscope.

Cells were transfected using Turbofectin (Origene, Herford, Germany) and cultivated for 24 h. Every transfection experiment included a negative control using transfection of the green fluorescent protein (GFP) and a non-mutated wildtype positive control. Transfection efficiency was assessed using fluorescence microscopy of PEDS1-deficient HAP1 cells transfected with GFP as follows. 48 h post transfection cell nuclei were counterstained with 1 µg/ml Hoechst 33342 (Sigma-Aldrich, Vienna, Austria) for 10 min at 37 °C. With a Leica DM IL LED inverted fluorescence microscope, fluorescent pictures of 4–6 areas per well of 6 parallel wells were taken (all 43 pictures were taken with the following filters: excitation 450–490 nm, emission 500–550 nm for GFP; excitation 381–393 nm, emission 417–477 nm for Hoechst 33342). Pictures were evaluated by Cell Profiler 2.2.1 [17 (link)] yielding a transfection efficiency of 4.15 ± 1.86% (mean ± SD, N = 43). Despite low transfection efficiency, we processed the cells further without an enrichment step for transfected cells, but with very sensitive detection methods for recombinant protein (western blot for a 6xmyc tag), and PEDS1 activity (fluorescence precursor labeling of the cells and detection of fluorescent newly formed plasmalogen).