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Alexa fluor 594 conjugated isolectin gs ib4

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Alexa Fluor 594-conjugated isolectin GS-IB4 is a fluorescent-labeled lectin that binds specifically to α-D-galactose and N-acetyl-α-D-galactosamine residues. It is commonly used as a marker for endothelial cells and microglia in various research applications.

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Lab products found in correlation

Lsm 880, by Zeiss (2 mentions) Fv1000, by Olympus (1 mentions) Image pro plus, by Media Cybernetics (1 mentions) Matrigel basement membrane matrix, by BD (1 mentions) Egm 2 bulletkit, by Lonza (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Anti p16ink4a antibody, by Abcam (1 mentions) Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Vectashield, by Vector Laboratories (1 mentions) P enos, by Cell Signaling Technology (1 mentions) Neutral buffered formalin, by Merck Group (1 mentions) Ve cadherin, by BD (1 mentions) Alexa fluor 488 conjugated secondary igg, by Thermo Fisher Scientific (1 mentions) P c kit, by Cell Signaling Technology (1 mentions) Anti alpha smooth muscle actin α sma antibody, by Abcam (1 mentions) Fitc conjugated dextran, by Thermo Fisher Scientific (1 mentions) Zen intellesis, by Zeiss (1 mentions) C3630, by Merck Group (1 mentions)

Close spelling variants of this product

Alexa Fluor 594 (2045 citations) Alexa 594 (538 citations) Isolectin GS-IB4 (51 citations) Alexa Fluors (21 citations) GS-IB4 (9 citations) Alexa Fluor® 594 conjugated isolectin GS‐IB4 antibody (2 citations)

8 protocols using alexa fluor 594 conjugated isolectin gs ib4

1

RPE-Choroid-Sclera Complex Analysis Post Laser

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On days 3, 7, and 14 after laser photocoagulation, RPE-choroid-sclera complexes were surgically isolated, fixed with 4% paraformaldehyde for 1 h, and then incubated with a blocking buffer containing PBS, 0.5% Triton X-100, and 5% bovine serum albumin for 4 h at 4°C. The complexes were then incubated with Alexa Fluor®-594 conjugated isolectin (GS-IB4; Invitrogen, Carlsbad, CA, USA). The CNV lesions were imaged with a confocal microscope (FV 1000; Olympus), and fluorescent signals were quantified (Image-Pro Plus; Media Cybernetics, Inc., Rockville, MD, USA) [9 (link)].
Wang C., Zhang R., Zhang Q., Jin H., Wei C., Wu C., Mei L., Liu Y, & Zhang P. (2020). Cytokine Profiles and the Effect of Intravitreal Aflibercept Treatment on Experimental Choroidal Neovascularization. Ophthalmic Research, 65(1), 68-76.
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2

Endothelial Tube Formation Assay

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For the tube formation assay, the cells separated by MACS were plated on 4-well slides (Iwaki Glass, Tokyo, Japan) coated thickly with BD Matrigel Basement Membrane Matrix (250 μL, Becton-Dickinson Co., Franklin Lakes, NJ) at 3 × 104 cells/cm2, following culture for 48 hours in EBM-2 medium with EGM-2 Bulletkit (LONZA, Basel, Switzerland). After the culture, cells were fixed with 4% PFA and stained with Alexa Fluor 594 conjugated isolectin GS-IB4 (Invitrogen) and DAPI (Merck). The tube formations were assessed with a fluorescence microscope (BX51, Olympus Optical) and images were obtained by a CCD camera (DP70, Olympus Optical).
Himeno T., Kamiya H., Naruse K., Cheng Z., Ito S., Shibata T., Kondo M., Kato J., Okawa T., Fujiya A., Suzuki H., Kito T., Hamada Y., Oiso Y., Isobe K, & Nakamura J. (2015). Angioblast Derived from ES Cells Construct Blood Vessels and Ameliorate Diabetic Polyneuropathy in Mice. Journal of Diabetes Research, 2015, 257230.
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3

Immunolabeling of Retinal Vasculature

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The isolated retinal vasculature was snap frozen in optimal cutting temperature (OCT) solution. Cryostat sections (10 μm) were obtained, mounted on glass slides and permeabilized with 1% Triton for 10 min and blocked in 10% normal goat serum for 1 h. Sections were then incubated with anti-p16INK4A antibody (Abcam, Cambridge, MA, USA; Cat. # ab189034; 1:50) and Alexa Fluor 594-conjugated isolectin GS-IB4 (Invitrogen, Carlsbad, CA, USA; Cat. # I21413; 1:200) at 4 °C overnight. On the next day, the sections were washed three times with phosphate-buffered saline (PBS) and were then incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA; Cat. # A11034; 1:400) that was washed in PBS, and covered with mounting medium containing 4′,6′-diamino-2-phenylindole (DAPI) for counterstaining (Vectashield; Vector Laboratories, Burlingame, CA, USA; Cat. # H-1200). Images from three to four samples per group were taken using Zeiss Axioplan2 Imager Microscope (Zeiss, San Diego, CA, USA). The results were analyzed by measuring the fluorescence intensity of p16 immunolabeling using ImageJ software.
Shosha E., Xu Z., Narayanan S.P., Lemtalsi T., Fouda A.Y., Rojas M., Xing J., Fulton D., Caldwell R.W, & Caldwell R.B. (2018). Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1. International Journal of Molecular Sciences, 19(4), 1215.
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4

Retinal Angiogenesis and Vascular Markers

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Retinas were fixed in 10% neutral buffered formalin (Sigma), embedded in paraffin, and sectioned. After quenching endogenous peroxidase activity and blocking with 10% normal horse serum, the sections were incubated with primary IgG against phospho-cKit (p-cKit; Cell Signaling Technology), p-eNOS (Cell Signaling Technology), or CD31 (DakoCytomation, Carpinteria, CA, USA), followed by the appropriate fluorescence-conjugated secondary IgG. For whole-mount retina staining, samples were blocked with 5% bovine serum albumin and 5% normal donkey serum in 0.5% Triton X-100, then stained with a primary IgG against VE-cadherin (BD Biosciences). The whole mounts were incubated with the appropriate Alexa Fluor 488conjugated secondary IgG (Invitrogen) and further stained with Alexa Fluor 594-conjugated isolectin GS-IB4 (Invitrogen) overnight. The number of sections examined ranged from 6 to 10 per group.
Kim S.R., Im J.E., Jeong J.H., Kim J.Y., Kim J.T., Woo S.J., Sung J.H., Park S.G, & Suh W. (2016). The cKit Inhibitor, Masitinib, Prevents Diabetes-Induced Retinal Vascular Leakage. Investigative ophthalmology & visual science, 57(3).
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5

Quantifying Muscle Capillary Density

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Ischemic gastrocnemius muscle and soleus muscle were fixed with 4% paraformaldehyde and embedded with paraffin. Paraffin sections were cut at 5 μm and stained with Alexa Fluor® 594 conjugated isolectin GS-IB4 (Thermo Scientific, Waltham, MA) to evaluate the capillary density. The number of capillaries were calculated in randomly selected fields for a total of 20 different fields ( × 40 magnification) per section and 3 sections per animal. The capillary density was expressed as capillary number per muscle fiber.
Dai X., Wang K., Fan J., Liu H., Fan X., Lin Q., Chen Y., Chen H., Li Y., Liu H., Chen O., Chen J., Li X., Ren D., Li J., Conklin D.J., Wintergerst K.A., Li Y., Cai L., Deng Z., Yan X, & Tan Y. (2022). Nrf2 transcriptional upregulation of IDH2 to tune mitochondrial dynamics and rescue angiogenic function of diabetic EPCs. Redox Biology, 56, 102449.
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6

Quantifying EPC Homing and Angiogenesis

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To examine the homing and incorporation of EPCs in the ischemic muscle, ischemic gastrocnemius muscle and/or soleus muscle were embedded in OCT medium for frozen section. The GFP positive EPCs were counted in randomly selected fields for a total of 20 different fields (×40 magnification) per section and 3 sections per animal. The EPC incorporation was expressed as the percentage of GFP positive capillaries.
The extent of angiogenesis and arteriogenesis at day 28 post ischemic surgery was assessed by measuring capillary density in gastrocnemius muscle and/or soleus muscle using isolectin B4 staining, and arteriole area in adductor muscle. Ischemic gastrocnemius muscle, soleus muscle and/or adductor tissues were fixed with 4% paraformaldehyde and embedded with paraffin. Paraffin sections were cut at 5 μm and stained with Alexa Fluor® 594 conjugated isolectin GS-IB4 (Thermo Scientific, Waltham, MA)or anti-alpha smooth muscle actin (α-SMA) antibody (Abcam) to evaluate the capillary density and arteriole area respectively. The number of capillaries or arteriole area were calculated in randomly selected fields for a total of 20 different fields (×40 magnification) per section and 3 sections per animal. The capillary density was expressed as capillary number per muscle fiber, and the arteriole area was expressed as α-SMA positive arteriole area per fiber and normalized by control.
Dai X., Yan X., Zeng J., Chen J., Wang Y., Chen J., Li Y., Barati M.T., Wintergerst K.A., Pan K., Nystoriak M.A., Conklin D.J., Rokosh G., Epstein P.N., Li X, & Tan Y. (2017). Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia. Circulation research, 120(5), e7-e23.
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7

Retinal Vascular Leakage Quantification

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FITC-conjugated dextran (70 kDa, 0.1 mg/g; Thermo Fisher Scientific; D1822) was injected retro-orbitally and allowed to circulate for 1 hour. Mice were then euthanized, and eyes were enucleated. Eyes were lightly fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, and retinas were dissected and flat-mounted. Whole-mounted retinas were imaged using confocal microscopy (LSM 880; Zeiss, Oberkochen, Germany). The fluorescence intensity of FITC-dextran was analyzed with Zeiss microscope software ZEN Intellesis to segment and quantify vascular leakage from retinal vessels based on previous protocols (60 ). Retinas were costained with Alexa Fluor 594–conjugated isolectin GS-IB4 (Thermo Fisher Scientific; I21413).
Wang Z., Liu C.H., Huang S., Fu Z., Tomita Y., Britton W.R., Cho S.S., Chen C.T., Sun Y., Ma J.X., He X, & Chen J. (2020). Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier. Science Advances, 6(35), eaba7457.
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8

Creatine Monohydrate Effects on Retinal Angiogenesis

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In OIR, mouse pups (C57BL/6J, male and female) were orally gavaged with creatine monohydrate (500 mg/kg; C3630; Sigma-Aldrich, St Louis, MO, USA) or PBS (control; 10010–023; Thermo Fisher Scientific) daily, from P12 to P16. Five mice each were allocated to the creatine treatment group and to the vehicle group. At P17, eyes were enucleated and retinas were stained with Alexa Fluor 594-conjugated isolectin GS-IB4 (10 μg/ml; I21413; Thermo Fisher Scientific), as described [24 (link)]. Then, retinas were visualised using anF epifluorescence microscope (AxioObserver.Z1; Carl Zeiss Microscopy, Jena, Germany). Mice weighing less than 5.0 g were excluded from this study [25 (link)]. At P17, the areas of retinal NV and vaso-obliteration (VO) were quantified as previously described [26 (link)].
Tomita Y., Cagnone G., Fu Z., Cakir B., Kotoda Y., Asakage M., Wakabayashi Y., Hellström A., Joyal J.S., Talukdar S., Smith L.E, & Usui Y. (2020). Vitreous metabolomics profiling of proliferative diabetic retinopathy. Diabetologia, 64(1), 70-82.
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