Axiovert 100m

TUNEL assay for the in situ detection of apoptosis was performed using the ApopTag® Red In Situ Apoptosis detection kit (Millipore, MA) according to the manufacturer's instructions. Frozen nude mouse prostate tumor (PC3) xenograft sections were also processed accordingly. Nuclei were counterstained with DAPI. Tissue sections were analyzed for apoptotic cells with localized fluorescence using an inverted fluorescence microscope (Zeiss Axiovert100M, Carl Zeiss, Germany).

Segments of the aortic arch and thoracic and abdominal aortas of 5-6 mice were frozen in liquid nitrogen and stored in −80°C. Later, the segments were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, Torrance, CA) for subsequent cryostat sectioning. The area analyzed as the aortic arch was the closest region to the aortic root, the thoracic aorta was the region posterior to the left subclavian artery, and the abdominal aorta was the closest to the diaphragm. Aorta sections were fixed in acetone for 40 min, permeabilized with PBS containing 0.1% Nonidet P-40 (Sigma-Aldrich, Saint Louis, MO) for 30 min, and blocked with albumin 1% for 1 h. Primary antibodies were incubated overnight at 4°C: anti-fibrillin-1 (sc-20084, Santa Cruz, Dallas, TX), anti-collagen I (ab90395, abcam, Cambridge, MA), and anti-collagen III (ab7778, abcam, Cambridge, MA). After incubation with fluorescein-labeled secondary antibodies (1 h at 37°C), slides were mounted with PBS containing glycerol (1 : 1, v/v) and DAPI (Invitrogen, 10 μg/mL). Images were obtained under 400x magnification on a Zeiss Axiovert 100M scanning confocal microscope and Axiovision software (Carl Zeiss, Jena, Germany). For expression measurements, we calculated the percentage between the positively marked area for each protein and the total tissue area.

TUNEL assay was performed using the Dead End™ fluorometric TUNEL assay kit. The TUNEL assay for in situ detection of apoptosis was performed by using the Dead End™ Fluorometric TUNEL System assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. H460 and A549 cells were treated with D1399 at concentrations of 0.5 × IC₅₀, 1.0 × IC₅₀, and 1.5 × IC₅₀ for 24 h. Following, cells were fixed in 4% paraformaldehyde solution at 4 °C for 25 min. Fixed cells were washed three times with 1 × PBS for 5 min and then permeabilized in 0.1% Triton X-100 for 15 min. After rinsing the slides with 1 × PBS, cells equilibrated in an equilibration buffer for 10 min and treated terminal deoxynucleotidyl transferase (TdT) was stored away from light for 1 h at 37 °C to label with fluorescein-12-dUTP. After a wash with 2 × SSC for 15 min at room temperature, the cells were treated with 1 mg·mL⁻¹ DAPI solution for 15 min. The slides were observed by fluorescence microscopy with an inverted microscope (Zeiss Axiovert100M, Carl Zeiss, Germany). A total of ten randomly chosen microscopic fields, including green fluorescence of apoptotic cells, were captured and calculated. Experiments were performed in triplicate.

In situ reactive oxygen species microfluorotopography of the LV was performed with dihydroethidium (DHE, Invitrogen, Carlsbad, CA). LV paraffin sections were deparaffinized and incubated in PBS Tween 1% for 30 minutes at room temperature. Sections were incubated with 5 μM DHE for 60 minutes at 37°C. Images were detected in a Zeiss Axiovert 100M scanning confocal microscope and Axiovision software (Carl Zeiss, Jena, Germany). Parallel reading of images was performed with identical laser acquisition settings. Quantitative analysis of fluorescent images of SEN and INT areas was performed with an image analysis system (Leica Q500 iW; Leica Imaging Systems, Cambridge, UK) under 400x magnification [15 (link)].

Cell apoptosis was detected with In Situ Cell Death Detection Kit (R&D Systems, USA), according to the manufacturer's protocol. Cells (1 × 10⁵ cells/well) were seeded in 24‐well plates and were treated. Next, cells were first fixed in 4% paraformaldehyde at 4°C for 30 minutes and permeabilized in 0.1% Triton X‐100, and treated with fluorescein‐12‐dUTP. Finally, the fluorescence was detected using fluorescence microscopy (Zeiss Axiovert 100 M, Carl Zeiss, Germany).

Apoptotic DNA fragmentation in TAMs induced by rhVEGI‐251 was examined using a DeadEnd™ Fluorometric TUNEL System kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.²² In brief, cells (at a density of 1 × 10⁵ cells/well) were seeded on coverslips in 24‐well flat‐bottom plates and treated with 2, 4 or 8 U rhVEGI‐251 for 24 hours. Following treated with rhVEGI‐251, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 30 minutes. Fixed cells were then washed with PBS for three times, permeabilized in 0.1% Triton X‐100 on ice for 2 minutes and labelled with fluorescein‐12‐dUTP for 1 hour by using terminal deoxynucleotidyl transferase at 37°C in a humidified chamber. After three further rinses with PBS, cell nuclei were double‐stained with propidium iodide (PI) at concentration of 1 μg/mL at room temperature for 15 minutes. The localized green (fluorescein‐12‐dUTP) or red (PI) fluorescence of apoptotic cells was detected by fluorescence microscopy (Zeiss Axiovert 100M, Carl Zeiss, Germany). Ten randomly chosen microscopic fields were imaged. Experiments were performed in triplicate.