Real-time monitoring Cy5.5-labeled nanoparticles (Cy5.5-PLGA) in retinal blood vessels.
Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)[2].
1. Freshly prepared suspensions of Cy5.5-PLGA (resuspended in 0.5 ml 1% poloxamer 188, vortexed gently and left for 30 min at ambient temperature before use) is administrated intravenously (b>0.5 mL).
2. Anesthetize the rats, treat the eyes with Neosynephrine-POS 5% to relax the iris, and Vidisic eye gel is applied to protect the eye from drying out and used as immersion medium for the contact lens as well.
3. Fix the rats under a confocal scanning microscope with the eye positioned in working distance underneath the objective lens, and a cannula is inserted into the tail vein.
4. Observe the fluorescence in the retina, and capture the images at different time points (b>0, 1, 3, 5, 15, 30, 60, 90 min).
Note: The rats are kept on the heating plate during all the in vivo imaging process.
5. After in vivo real-time imaging, rats were euthanized with an overdose of aforementioned anesthetic and the eyeballs were enucleated and placed into cooled HEPES buffered solution (135 mM NaCl, 5 mM NaOH, 2.5 mM KCl, 7 mM MgCl2, 10 mM HEPES, 10 mM glucose; pH7.4).
6. Remove the anterior segment of eye and vitreous body, separate whole retina carefully, flat on the modified culture plate.
7. Incubate the whole mounts with0.1 mg/mL Hoechst Hoechst 33342 (HY-15559) in HEPES solution for 20 min for nuclei staining.
8. Fix flat mount retina with 4% paraformaldehyde solution for 20 min and wash with HEPES solution.
9. Capture the images immediately with microscope after preparation of retinal flat mount.
