Fv1000 confocal laser microscope

After in utero electroporation, organotypic coronal slices (250 μm thick) from the interventricular foramen were prepared with a microtome, placed on an insert membrane (pore size, 0.4 μm; Millipore, Bedford, MA), mounted in agarose gel and cultured. The dishes were then mounted in an incubator chamber (5% CO2 and 40%O2, at 37 °C) fitted onto an FV1000 confocal laser microscope (Olympus, Tokyo, Japan), and the primary somatosensory cortex was examined as described35 (link). Approximately 8–15 optical Z sections were acquired automatically every 8 to 15 min for 24 h, and about 10 focal planes (~50 μm-thickness) were merged to visualize the entire shape of the cells.

Cells were seeded onto coverslips in 24‐well plates overnight and exposed to different experimental conditions, and then, the cells were fixed in 4% (w/v) paraformaldehyde/PBS for 20 minutes. After fixation, the cells were subjected to proteinase K digestion for 1 minute, permeabilized with 0.1% Triton X‐100 for 5 minutes and blocked with bovine serum albumin for 30 minutes. The cells were incubated with primary antibody overnight at 4°C and then stained with FITC‐conjugated secondary antibodies (1:500 dilution) for 30 minutes in the dark. The cells were then treated with Hoechst 33342/H₂O (1 μg/mL) for 2 minutes. For staining of mitochondria and lysosomes, the cells were incubated with MitoTracker RED (200 nmol/L, 30 minutes), LysoTracker Green (50 nmol/L, 60 minutes) or DQ Red BSA (10 µg/mL, 2 hours). After rinsing with PBS, images were acquired by using an Olympus FV1000 confocal laser microscope (Olympus Corporation, Japan).

Fetal arteries, including the DA and the aorta, were removed from the thoracic cavity. Fetal arterial tissues and three-dimensional (3D) vascular multilayers were fixed in a zinc-based fixative for 36 h and embedded in paraffin [24 (link)]. The paraffin-embedded tissue sections (thickness, 8 μm) were heated at 59°C overnight, deparaffinized in xylene, and rehydrated in graded alcohol baths. Dissolved DQ gelatin (20 μg/ml) (Invitrogen, Waltham, MA, USA) was added on the tissue sections, and they were then incubated for 2 h in a dark, humid chamber. The sections were then rinsed with double-distilled water and fixed in 4% neutral-buffered formalin for 10 min in the dark. Sections were then rinsed twice in phosphate-buffered saline (PBS), and DAPI (4′, 6-diamidino-2-phenylindole) (Invitrogen, Waltham, MA, USA) was added to stain the nuclei. After 20 min, sections were rinsed in PBS and mounted using aqueous mounting medium. To verify the contribution of matrix metalloprotease (MMP), control slides were pre-incubated with ethylenediaminetetraacetic acid (EDTA, 20 mM) for 1 h. EDTA (20 mM) was also added to the substrate. Fluorescence was detected using a FV1000 confocal laser microscope (Olympus, Tokyo, Japan).

Antibodies against α-tubulin (Sigma, T6074) and the secondary antibodies Alexa Fluor 594 (Life Technologies Corp.) were employed in immunofluorescence staining. Microscopic analysis that clearly reflecting changes of axonal length were captured using an Olympus FV1000 confocal laser microscope.

Cells were fixed in 4% (w/v) paraformaldehyde (PFA)/PBS for 20 minutes and then permeabilized with 0.1% Triton X‐100 for 15 minutes. After blocking with bovine serum albumin for 30 minutes, cells were incubated with primary antibody overnight at 4°C. Cells were then incubated with FITC/Texas Red‐conjugated secondary antibodies (Proteintech) at room temperature for 1 hour. The images were acquired using an Olympus FV1000 confocal laser microscope.