Eyes were removed from neonates at postnatal day 5 (P6) and prefixed in 4% paraformaldehyde (4% PFA) for 15 min at room temperature. Dissected retinas were blocked overnight at 4 °C in TNBT (0.1 M Tris-HCl, 150 mM NaCl, 0.2% blocking reagent (PerkinElmer) supplemented with 0.5% TritonX-100). After washing, the retinas were incubated with Isolectin GS-IB4, Alexa Fluor® 488 Conjugate (ThermoFisher #I21411) in Pblec (1 mM MgCl2, 1 mM CaCl2, 0.1 mM MnCl2, 1% Triton X-100 in PBS) for 2 h at RT. Retinas were washed 6 times, for 10 min in PBS, fixed briefly for 5 min in 4% PFA, washed twice in PBS and mounted in fluorescent mounting medium (DAKO mounting media #CS703). Low- and high-magnification images were acquired using fluorescent (Nikon 80i Nikon Ti-E Eclipse inverted microscope) and confocal (ZEISS LSM710 laser scanning confocal) microscopes. ImageJ was used to measure distance from center to vascular edge (VE) and center to retina edge (RE). Vascular progression is reported as VE/RE ratio. Biological CMM Analyzer software 16 was used for quantification of branch points per image63 (link).
Lsm710 laser scanning confocal
Manufactured by Zeiss
The LSM710 laser scanning confocal microscope is a versatile imaging system designed for high-resolution fluorescence and reflected light microscopy. It employs a focused laser beam to scan the sample, allowing for the acquisition of optical sections with improved contrast and resolution compared to conventional widefield microscopy.
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2 protocols using lsm710 laser scanning confocal
1
Retinal Vasculature Quantification Assay
2
Fluorescence Imaging of Brain Sections and EV Uptake
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For in vivo sections, the brains were removed, kept in 4% PFA (EMS, 15714) overnight and 30% sucrose for 48 h, and then cryosectioned at 40-μm thickness. Slides were then mounted with Antifade Mounting Medium with DAPI (Vector Labs, H-1200-10). Fluorescence microscopy images were acquired on a Keyence (Itasca) microscope and processed using ImageJ2 v2.3.0 (RRID:SCR_003070 ).
For in vitro EV uptake experiments, bone marrow-derived macrophages (BMDM) were exposed to EVs for 24 h, washed with PBS for three times, and fixed using 100% ice-cold methanol for 10 min. After fixation, cells were rinsed twice in PBS for 5 min each. Blocking was achieved by using 5% BSA and 0.1% Tween 20 (Sigma Aldrich, P1379-25ML) in PBS (PBS-T) for 4 h. Cells were then incubated with the anti-mouse F4/80 Antibody conjugated to Alexa Fluor 488 (BioLegend, #123119, RRID:AB_893491 ) for 1h at room temperature. Cells were rinsed three times in PBS-T for 5 min each. Coverslips were transferred to microscope slides (Fisherbrand) on a droplet of mounting medium containing DAPI (Vectashield; Vector Labs, H-1200-10). Fluorescence microscopy images were acquired on a Keyence (Itasca) and LSM710 Laser Scanning Confocal (Zeiss) microscope and processed using ImageJ2 v2.3.0 (RRID:SCR_003070 ).
For in vitro EV uptake experiments, bone marrow-derived macrophages (BMDM) were exposed to EVs for 24 h, washed with PBS for three times, and fixed using 100% ice-cold methanol for 10 min. After fixation, cells were rinsed twice in PBS for 5 min each. Blocking was achieved by using 5% BSA and 0.1% Tween 20 (Sigma Aldrich, P1379-25ML) in PBS (PBS-T) for 4 h. Cells were then incubated with the anti-mouse F4/80 Antibody conjugated to Alexa Fluor 488 (BioLegend, #123119, RRID:
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