Alexa fluor 647 dye

Dulbecco’s modified Eagle’s medium (DMEM), PBS, penicillin/streptomycin, l-glutamine, fetal bovine serum (FBS), Aqua Dead Cell Stain Kit, Lipofectamine 2000, TRIzol, and collagenase type IV were purchased from Thermo Fisher Scientific (Waltham, MA, United States). The RBC lysis buffer was purchased from Solarbio (Beijing, China). siRNA-targeting mouse CD47 mRNA (antisense strand, 5’-UGGUGAAAGAGGU-CAUUCCdTdT-3’) and negative control siRNA with a scrambled sequence (antisense strand, 5’-ACGUGACACGUUCGGAGAAdTdT-3’) were synthesized by Suzhou Biosyntech Co. Ltd. (Suzhou, China). Anti-CD47 antibody was purchased from Santa Cruz Biotechnology (Texas, United States). The Click-iT Plus TUNEL Assay was performed for in situ apoptosis detection; the Alexa Fluor 647 dye was purchased from Thermo Fisher Scientific (MA, United States).

All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at UConn Health. Wild-type C57BL/6 mice were obtained from the National Institute on Aging (NIA) and maintained in a pathogen-free facility at 23–24 °C under a 12-h light, 12-h dark regimen with free access to a standard mouse diet (Teklad global 18% protein, Envigo #2918, Indianapolis, IN) and water. Dasatinib (LC Laboratories, Woburn, MA) and quercetin (Sigma-Aldrich, St Louis, MO) were dissolved in DMSO for in vitro treatment. Scaffold discs (3.5 mm diameter × 0.5 mm thickness) were purchased from Healos HA (DePuy Orthopaedics, Raynham, MA). Alexa Fluor647 dye, ELF^®97 phosphatase substrate, and cryomatrix™ were purchased from Thermo Fisher Scientific (TWaltham, MA, USA). Fast Red-TR and Naphthol AS-MX Phosphate was obtained from Sigma-Aldrich. All other materials were purchased from Thermo Fisher Scientific.

For DiI fills, embryos or larvae were fixed at relevant developmental stages in 4% PFA at 2 hr at RT, or overnight at 4°C. Specimens were then carefully placed in a small petri dish with a sylgard-coated bottom containing PBS and secured with the eye facing up using a Tungsten minuten pin. The eye was carefully removed from the orbit with sharp forceps and DiI (D3911, Invitrogen) was applied to the tissue at the corner of the orbit, where the oculomotor and trochlear nerves enter, using a blunt microinjection needle held by a micromanipulator. DiI was left to bind to the tissue for 3–5 min, after which embryos were stored overnight in PBS at 4°C. The following day, brains were dissected from whole larvae and mounted for imaging. For live retro-orbital dye fills, ocular motor nuclei were retrogradely labelled as previously described (Greaney et al., 2017 (link)). Crystallised fluorescently-conjugated Alexa Fluor 647 dye (10,000 molecular weight, Thermo Fisher D-22914) was applied unilaterally to the orbit of anesthetised fish at four dpf and the side of incision and dye application was recorded for each larva. Larvae were returned to the incubator at 28.5°C to recover overnight before imaging at 5 dpf.

The iDISCO (ace) procedure was performed according to the previously described protocol (Liu et al., 2020 (link)). The VE-cadherin antibody conjugated with Alexa Fluor™ 647 (VE-cadherin antibody, Biolegend, #138002. Alexa Fluor™ 647 dye, Thermo Fisher, #A20006) was diluted 1:1000 for use. After immunolabeling with Alexa Fluor dye-conjugated VE-Cadherin antibody, the tissues were washed directly with PBS/0.1% Tween 20/heparin (10 μg/ml) at room temperature for 24 h following the protocol described. The lung tissues processed by the iDISCO (ace) procedure were imaged on an Andor Dragonfly 200 Confocal Imaging System, with a ×10 objective, a step size of 8μm, 250 ms exposure time. About 1200-μm-thick optical stack of signal were acquired for each lung tissue. Imaris (version 9.9) was used to reconstruct the image stacks obtained from the confocal imaging to perform whole-tissue 3D assessment of the vasculature. AngioTool (version 0.6a) was used for vessels percentage area and total number of junctions analysis (Zudaire et al., 2011 (link)).

RN2N was covalently conjugated with Alexa Fluor 647 dye (Thermo Fisher Scientific) in PBS with 0.1 M sodium bicarbonate, as described previously^{14 (link)}. The labelled antibodies were then separated from free dye using a Superdex 200 10/300 column (GE Healthcare) equilibrated in 1 X PBS, pH 7.4, at 0.5 mL/min, and the degree of labeling was determined. Antibody-Alexa Fluor 647 conjugates were then concentrated to the desired injection volumes using an Amicon® Ultra filter (Millipore).