Radioimmunoprecipitation assay buffer

The rabbit eyes were immediately immersed in 100% isopropyl alcohol, then directly transferred into liquid nitrogen to preserve the post-SCS injection distribution of particles. The completely frozen eyes were cut by a razor from the optic nerve to the limbus to make eight symmetric petals. Then, the cut petals were peeled off and splayed like a flower to observe the particle distribution in the eye from the chorioretinal side. The ocular petals were imaged by a camera (Cannon 60d; Canon, Melville, NY, USA) to generate bright field and fluorescence images (λex = 580/ λem = 605). A green LED light (Bluewind Multicolor RGB; HitLight, Baton Rouge, LA, USA) was used as a light source and an optical bandpass filter (610 ± 10 nm; Edmunds Optics, Barrington, NJ, USA) was mounted on the camera to get the desired fluorescence images.
The SCS petals were divided and sliced according to the distance from the limbus (0–3, 3–6, 6–9, and >9 mm) to calculate the particle distribution. To extract the particles, the petals were lysed and sonicated in radioimmunoprecipitation assay buffer (Abcam, Cambridge, UK) to disrupt the tissues. Then, the fluorescent signals of the extracted-particle solutions were measured by a plate reader (Synergy Microplate Reader, Winooski, VT, USA).

Whole-cell lysates were made from homogenized isolated pulmonary artery samples with radio-immunoprecipitation assay buffer (Abcam, UK) containing a protease inhibitor cocktail (Roche, UK). Protein concentrations were determined through a Direct Detect Spectrometer (Merck, Germany). 20 µg protein from each sample was resolved by SDS-PAGE and transferred to nitrocellulose membranes for Western blotting with antibodies specifically recognizing human AMPK and phosphorylated AMPKα (p-AMPK) (Cell Signaling Technology, US), as well as eNOS, phosphorylated eNOS (p-eNOS), ERA, ERB and GAPDH (Abcam, UK). Immune complexes were visualized using horseradish peroxidase-conjugated secondary antibody with enhanced chemiluminescence on a BioRad Chemidoc MP system.

The silencing effects of siRNA targeting the LIMD1 gene were assessed by western blot analysis. The cells were cultured in a six-well plate at a density of 2.0×10⁴ cells/well for 48 h, harvested, washed twice with ice-cold phosphate-buffered saline and then lysed in radioimmunoprecipitation assay buffer (Abcam, Cambridge, UK). The cell lysates were briefly sonicated (Sonicator Q700; Qsonica, LLC, Newton, CT, USA) and kept in ice-water for 30 min. The protein concentrations were determined using a BCA Protein Assay kit (Bio-Rad, Hercules, CA, USA). The protein bands were visualized by chemiluminescence using enhanced chemiluminescence plus western blotting reagent (GE Healthcare, Little Chalfont, UK), followed by exposure to Fujifilm LAS-1000 equipment (Fujifilm, Tokyo, Japan). The parallel membranes were incubated with 1:10,000 rabbit anti-human monoclonal antibodies against β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and horseradish peroxidase-coupled rabbit anti-mouse monoclonal secondary antibody (Innova Biosciences, Ltds., New York, NY, USA).