At several time points after suprachoroidal injection of 3 μl of NPs containing 1 μg of pEGFP-N1, rats were euthanized and eyes were frozen in optimal cutting temperature embedding compound (Miles Diagnostics, Elkhart, IN). Frozen sections (10 μm) were fixed in 4% paraformaldehyde, and nonspecific binding was blocked by a 30-min incubation in 8% normal rabbit serum at 25°C. The sections were incubated with a polyclonal antibody (1:300) against EGFP conjugated with Alexa Fluor 594 (A-21312, ThermoFisher, Waltham, MA) at 23°C for 2 hours. After washing with PBS containing 0.05% Tween 20, slides were counterstained with Hoechst 33258 (861405, Sigma-Aldrich, St. Louis, MO) and viewed with a Nikon fluorescence microscope. Ocular sections from rats that had been given a suprachoroidal injection of NPs containing VEGF expression plasmid or RPE whole mounts from rho/VEGF mice were stained with fluorescein isothiocyanate–labeled (FL-1201, Vector Laboratories, Burlingame, CA) or DyLight 594–labeled Griffonia simplicifolia lectin (DL-1207, Vector Laboratories), which selectively stains vascular cells as previously described (38 (link)).
Dl 1207
Manufactured by Vector Laboratories
Sourced in United States
DL-1207 is a laboratory instrument designed for protein analysis. It is capable of detecting and quantifying proteins through a colorimetric assay.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by Roche (2 mentions)
Nb100 304, by Novus Biologicals (2 mentions)
Ab5539, by Merck Group (2 mentions)
Sc 188, by Alomone (2 mentions)
Acc 030, by Novus Biologicals (2 mentions)
Nbp1 49461, by Novus Biologicals (2 mentions)
Mab 1914p, by Vector Laboratories (2 mentions)
Eclipse 80i microscope, by Nikon (2 mentions)
Fl 1201, by Vector Laboratories (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Ab92513, by Abcam (1 mentions)
Sa5 10038, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by Zeiss (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
5 protocols using dl 1207
1
Suprachoroidal Nanoparticle Delivery and Imaging
2
Dorsal Root Ganglion and Spinal Cord Analysis
3
Retinal Development Imaging Protocol
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Eyes were collected from neonatal mice on P6, P15, or P60 and then fixed in 4% PFA for 2 hours at room temperature. Retinas were isolated and permeabilized in 0.3% TritonX-100 and 1% BSA/PBS overnight at 4°C. Followed by incubation with Isolectin B4 or primary antibody in 0.3% TritonX-100 and 5% donkey serum in 5% BSA/PBS overnight at 4°C. For Erg1 staining, the retinas were washed several times in PBS and incubated with fluorescent secondary antibodies. Reagents used were as follows: Dylight 594 isolectin-B4 (DL1207, Vector, 1:200 dilution), rabbit anti-ERG (ab92513, Abcam, 1:200 dilution), IgG (H+L) cross-adsorbed donkey anti–rabbit DyLight 488 (SA5-10038, Invitrogen, 1:500 dilution), phospho-Histone H3 (Ser10) mouse mAb (9706, CST, 1:200 dilution), and Alexa Fluor 647 conjugate (4410, CST, 1:500 dilution). Imaging was performed using a Zeiss Axio-Imager LSM-800 confocal microscope.
4
Dorsal Root Ganglion and Spinal Cord Analysis
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Dorsal root ganglia (DRG) and spinal cords were harvested from injured mAtf3pro/RmGFP reporter mice perfused with ice-cold PBS followed by cold 4% paraformaldehyde (PFA). In addition, sciatic nerves were harvested distal to the crush in treated mAtf3pro/RmGFP reporter mice perfused with cold 4% PFA. Perfused tissues were post-fixed for 3 hr at 4°C, and cryoprotected with 30% sucrose in PBS overnight. DRG sections (10 μm), spinal cord sections (20 μm) and sciatic nerve sections (60 μm) collected, blocked and permeabilized with 1% Triton X-100 in blocking buffer (Roche Diagnostics) for 1 hr at RT. Sections were incubated with rabbit polyclonal antibody against ATF3 (Santa Cruz Biotech; sc-188; 1:1000), TRPV1 (Alomone; ACC-030; 1:1000), SCG10 (Novus; NBP1-49461; 1:2000), 53BP1 (Novus; NB100-304; 1:2000), chicken polyclonal antibody against NF200 (Millipore, AB5539; 1:2000), rat polyclonal antibody against Laminin-γ (Millipore; MAB-1914P; 1:1000), DyLignt_594 conjugated Isolectin B4 (VectorLab; DL-1207; 1: 200), or mouse monoclonal antibody against phospho-Histone H2A.X (Ser139)(γ-H2AX; gamma-H2AX) (Millipore; 05-636; 1:400) at 4°C overnight and then incubated with Alexa Fluor 568 goat antibody against rabbit IgG, chicken IgG or rat IgG for 1 hr at RT. Images were acquired using a Nikon Eclipse 80I Microscope.
5
Angiogenesis Visualization in Cardiac Tissue
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The cardiac tissue samples were embedded in paraffin and cut at a thickness of 4 μm. After dewaxing in water, repairing antigens and blocking, the sections were labeled with isolectin B4 (10 μg/mL, DL-1207, Vector laboratories, USA) and DAPI (1 μg/mL, D1306, Thermo Fisher Scientific, Waltham, MA, USA) for angiogenesis observation. The procedures were carried out in accordance with the manufacturer's instructions. Sections (6 sections per group, 5 fields per section) were observed with fluorescence microscopy (Olympus, Japan) and laser scanning confocal microscopy (Zeiss, Germany). in WT and DDAH1 KO mice.
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