Aliquots of epididymal and subcutaneous adipose were fixed in 4% PFA then washed in PBS. Adipose was blocked and permeabilized in 5% BSA, 0.3% Triton in PBS before incubating overnight with CD68-PE conjugate (Biolegend, Clone FA-11) and Isolectin GS-IB4 AF488 conjugate (Thermofisher) or CD31 AF488 (Biolegend, Clone MEC13.3) at 4°C. After a final wash, samples were mounted in a 1:1 solution of PBS: Glycerol and digital images were acquired using confocal microscopy (Nikon Instruments Incorporated, Model TE200-E2; 20X objective). Images were processed using ImageJ software.
Isolectin gs ib4 af488 conjugate
Manufactured by Thermo Fisher Scientific
Isolectin GS-IB4 AF488 conjugate is a fluorescently-labeled lectin derived from the seeds of the Griffonia simplicifolia plant. It binds specifically to terminal α-D-galactose and N-acetyl-α-D-galactosamine residues on cell surfaces.
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5 protocols using isolectin gs ib4 af488 conjugate
1
Adipose Tissue Immunostaining Protocol
2
In Vivo Hydrogel Implant Analysis
3
In Vivo Hydrogel Implant Analysis
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G*Power was first used to compute the required sample size. Using two-tailed t-test on previous SubQ data and high variance assumption for the tortuosity measurement, we found the required sample size to be within the range of 3 to 6. We choose to start with 6 samples (2 gel samples on each animal) per group.
All in vivo studies were conducted in compliance with the NIH Guide for Care and Use of Laboratory Animals and UCLA ARC standards. Gel implantation was performed as previously described59 (link). At day 7, the clips closing the incision were taken off and after 2 weeks, each mouse was injected with 100ul of 1mg/ml of isolectin GS-IB4-AF488 conjugate (ThermoFisher Scientific, #I21411) through the left external jugular vein before and sacrificed by isoflurane overdose. The implant hydrogels (total of 6 blank gels, 6 Fn9*10 gels, 6 Fn9(4G)10 gels) were then collected and fixed in 1% PFA for 16 hours at 4°C. Due to the variance in sample collection process, the membrane layers attached to the implants were intact only for 4 gel implant per condition. Thus, for confocal imaging, only those with intact membranes were analyzed.
All in vivo studies were conducted in compliance with the NIH Guide for Care and Use of Laboratory Animals and UCLA ARC standards. Gel implantation was performed as previously described59 (link). At day 7, the clips closing the incision were taken off and after 2 weeks, each mouse was injected with 100ul of 1mg/ml of isolectin GS-IB4-AF488 conjugate (ThermoFisher Scientific, #I21411) through the left external jugular vein before and sacrificed by isoflurane overdose. The implant hydrogels (total of 6 blank gels, 6 Fn9*10 gels, 6 Fn9(4G)10 gels) were then collected and fixed in 1% PFA for 16 hours at 4°C. Due to the variance in sample collection process, the membrane layers attached to the implants were intact only for 4 gel implant per condition. Thus, for confocal imaging, only those with intact membranes were analyzed.
4
Adipose Tissue Macrophage Immunofluorescence
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Aliquots of epididymal and subcutaneous adipose were fixed in 4% PFA then washed in PBS. Adipose was blocked and permeabilized in 5% BSA, 0.3% Triton in PBS before incubating overnight with an anti-CD68-PE conjugated antibody (Biolegend, Clone FA-11) and an Isolectin GS-IB4-AF488 conjugate (Thermofisher) at 4°C. After a final wash, samples were mounted in a 1:1 solution of PBS:Glycerol and digital images were acquired using confocal microscopy (Nikon Instruments Incorporated, Model TE200-E2; 20X objective). 40 μm Z-stacks with 2 μm step size were acquired with a 20x magnification power and processed using ImageJ software.
5
Aortic Ring Assay for Angiogenesis
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The aortic ring assay was performed as described previously.37 (link) Thoracic aortae were dissected from C57/BL6 mice, cleaned, and cut into rings. The cells were then serum-starved overnight. During serum starvation, the rings were transfected with a miR agonist cocktail or negative control. The next day, rings were embedded in the following extracellular matrices: rat-tail collagen type I (Millipore, Tokyo, Japan, 08-115); Medium 199 10× phenol red-free (Thermo Fisher); 140 mM NaHCO3, 1 M NaOH, and filtered and autoclaved water. After 24 h, immunofluorescence staining was performed using Isolectin GS-IB4 AF488 conjugate (Thermo Fisher, I21411). The samples were then scanned using a fluorescence microscope (Keyence). Quantitative analyses were performed using Adobe Photoshop CS software program (Adobe system).
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