Inverted fluorescence microscope

Thoracic aortas slices (5 μm thick) were deparaffinized and subjected to hematoxylin-eosin (HE) staining (Service Biological Technology Co., Ltd., Wuhan, China). Images of three microscopic fields in each slice were captured (magnification, ×400) under an inverted fluorescence microscope (Olympus, Tokyo, Japan), and the relative count of vascular smooth muscle cells (VSMCs) was measured by Image-Pro Plus 6.0 software, independent of the orientation, form or size of cell nucleus.
Thoracic aortas slices (5 μm thick) were deparaffinized and subjected to Masson staining (Service Biological Technology Co., Ltd., Wuhan, China). Images of three microscopic fields in each slice were captured (magnification, ×400) under an inverted fluorescence microscope (Olympus, Tokyo, Japan), and collagen deposition in the vascular wall was analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, United States). The ratio of collagen (blue) to the fixed area of the thoracic aorta was calculated as the result of semi-quantitative analysis of collagen deposition.

The cells were plated on crystal 6-well slice at the density of 5 × 10⁵/ml in the culture plates, and treated with Dex and different groups of exosomes for 24 h. The samples were washed thrice in PBS, fixed in 4% paraformaldehyde, and permeabilized for 15 min with 0.1% TritonX-100 in PBS. The 5% bovine serum albumin was used to block cells at 37 °C for 1 h, followed by washing with PBS, and culturing with primary antibodies against Col-I (1:200, abcam, USA), or overnight at 4 °C. The TRITC Phalloidin, Alexa Fluor^®488-labeled secondary antibodies (1:400, abcam, USA) and DAPI solution (1:10) were added in sequence for 20 min, 1 h and 5 min at room temperature respectively, followed by 3 times rinsing in PBS. The images were captured by an inverted fluorescence microscope (Olympus, Japan). As for histofluorescence, the sections were deparaffinized and hydrated and incubated with a mixture of EMCN (1:200, Invitrogen, USA) and CD31(1:200, Invitrogen, USA). After washed with PBS, sections were incubated with secondary antibodies: Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated (1:200, Abcam) at room temperature for 30 min. The nuclei were stained with DAPI solution. The images were captured by an inverted fluorescence microscope (Olympus, Japan).

When cells reached 70-80% confluence, they were fixed with 4% paraformaldehyde overnight at 4°C. The cells were then blocked and permeabilized for 1 h at 37°C in PBS with 2% bovine serum albumin (BSA; Thermo Fisher, USA) and 0.1% Triton X-100, followed by incubation with rabbit anti-nestin (Boster, China), rabbit anti-pax6 (Boster, China), rabbit anti-GFAP (Boster, China), rabbit anti-S100 (Boster, China), and rabbit anti-β-III tubulin (Applied Biological Materials Inc., Canada) at the appropriate dilution overnight at 4°C. After washing with PBS, the cells were incubated at 37°C for 2 h with appropriate secondary antibodies: CyTM3-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson, USA). Nuclei were counterstained with DAPI. Images were captured using an Olympus inverted fluorescence microscope (IX73, Olympus, USA).

AO/EB staining was performed to study cell apoptosis post transfection as described previously [40 (link),41 (link)]. Cells at a density of 1.2 × 10⁵ cells/well were plated into a 24-well plate and incubated overnight for attachment. Thereafter, complexes were added to the cells at their various binding ratios and incubated for 24 h. The cells were then washed twice with PBS, and 10 μL of AO/EB dye (100 μg/mL AO and 100 μg/mL EB in PBS) was added. Cells were stained at room temperature for 5 min. Thereafter, the dye was removed, and the cells were viewed under an Olympus inverted fluorescence microscope fitted with a CC12 fluorescent camera (excitation filter of 450–490 nm and a barrier filter of 520 nm) (Wirsam Scientific and Precision Eq. LTD., Johannesburg, South Africa) at X200 magnification. Cells were examined for morphological changes due to apoptosis. Apoptosis was represented as an index, calculated as shown below: