Alexafluor 568 conjugated isolectin b4

Manufactured by Thermo Fisher Scientific

Sourced in United States

AlexaFluor 568-conjugated Isolectin B4 is a fluorescently-labeled lectin that binds to terminal α-D-galactose residues on cell surfaces. It is commonly used as a histological marker to label microvascular endothelial cells and macrophages.

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Lab products found in correlation

Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions) Vectashield mounting medium, by Vector Laboratories (3 mentions) Lsm 800, by Zeiss (2 mentions) Imaris, by Oxford Instruments (2 mentions) Confocal laser scanning microscope, by Olympus (2 mentions) Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions) Fluoview fv1000 confocal microscopy, by Olympus (1 mentions) Fv1000, by Olympus (1 mentions) Fluoromount g, by Southern Biotech (1 mentions) α sma, by Thermo Fisher Scientific (1 mentions) Fitc conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions) α sma, by Olympus (1 mentions) α sma, by Cell Signaling Technology (1 mentions) Alexa 555, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)

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Isolectin B4 (39 citations)

8 protocols using alexafluor 568 conjugated isolectin b4

Visualizing Retinal Vasculature in Aged Mice

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After OCTA imaging of 10-month-old mice, they were euthanized and their eyes were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) at 4 °C overnight. Next, retinas were dissected, washed with PBS, blocked and permeabilized with PBS containing 5% normal goat serum (NGS) and 0.3% Triton-X-100 for 3 h. Subsequently, retinas were incubated with Alexa Fluor 568-Conjugated Isolectin B4 (Thermo Fisher Scientific, Waltham, MA) at 4 °C for 3 days. After washing, retinas were mounted and imaged by confocal microscopy (LSM 800, Carl Zeiss, Thornwood, NY).
Confocal images were paired with the corresponding position of prior OCTA scans of mice at 10 months of age. Moreover, confocal images taken at central and mid-peripheral retinas (Fig. 1A) were analyzed with AngioTool for vessel density. Data from each retinal quadrant was likewise averaged and included as one data point in statistical analysis.

Buscho S., Palacios E., Xia F., Shi S., Li S., Luisi J., Kayed R., Motamedi M., Zhang W, & Liu H. (2022). Longitudinal characterization of retinal vasculature alterations with optical coherence tomography angiography in a mouse model of tauopathy. Experimental eye research, 224, 109240.

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Visualizing Retinal Vasculature in Aged Mice

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Choroid and Retinal Flat Mount Imaging

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The entire ocular globes were enucleated and fixed in 4% paraformaldehyde for 1 h. Choroid and retinal flat mounts were obtained by carefully removing the cornea, iris and lenses. The remaining eyecups were subjected to four radial incision towards the optic nerve head for flat mounting. The neurosensory retina was detached by sectioning the optic nerve. The retinal pigment epithelium (RPE)/ choroid/sclera complexes were permeabilized overnight with tris‐buffered saline (0.5% BSA, 0.2% Tween‐20 and 0.1% Triton X‐100). After repeated washes, samples were incubated with a 10 mg/mL solution of 40, 60‐diamidino‐2‐phenylindole (DAPI) (1:500), 1 mg/mL solution of Alexa Fluor 568‐conjugated isolectin B4 (1:100) and 0.2 U/mL solution of Alexa Fluor 488‐conjugated phalloidin (1:100; Invitrogen‐Molecular Probes) overnight at 4℃ in blocking solution. Tissues were mounted onto glass slides and cover‐slipped. Images were captured with Olympus Fluoview FV1000 confocal microscopy at 20× magnification. CNV lesions were identified by their geographic location revealed by the IsolectinB4 (IB4) signal. Image J was used to dissect out the areas of hyperfluorescence for further analysis by blinded observers.

Zhang W., Dai L., Li X., Li Y., Hung Yap M.K., Liu L, & Deng H. (2020). SARI prevents ocular angiogenesis and inflammation in mice. Journal of Cellular and Molecular Medicine, 24(7), 4341-4349.

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Choroidal Flat Mount Technique

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Seven days after laser coagulation, 30 eyes (six eyes from three mice/each group) were subjected to choroidal flat mount. The eyes were enucleated and immediately fixed in 4% paraformaldehyde (Guoyao Group of Chemical Reagents, Beijing, China) in PBS (pH 7.3) for 1 h. Under a biopsy microscope, the anterior segments were wiped out, and the neurosensory retinas were detached and separated from the optic nerve head. The remaining eyecups were washed with cold immunochemistry (ICC) buffer (0.5% BSA, 0.2% Tween-20, and 0.1% Triton X-100) in PBS. A 1 mg/ml solution of Alexa Fluor 568 conjugated isolectin-B4 (1:100; Invitrogen-Molecular Probes, Eugene, OR) was prepared in ICC buffer. The eyecups were incubated with the fluorescent dyes above in a humidified chamber at 4 °C with gentle shaking for 4 h, followed by washing with cold ICC buffer. Four or five radial cuts were made toward the optic nerve head for flat mounting of the sclera/choroid/RPE complexes with the gel (Gel-mount; Biomedia Corp. Foster City, CA). The samples were covered and sealed for microscopic analysis.

Yan Z., Shi H., Zhu R., Li L., Qin B., Kang L., Chen H, & Guan H. (2018). Inhibition of YAP ameliorates choroidal neovascularization via inhibiting endothelial cell proliferation. Molecular Vision, 24, 83-93.

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Quantifying Choroidal Neovascularization Volume

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Eyes were first fixed in 4% paraformaldehyde (Electron Microscopy Sciences [EMS], Hatfield, PA) for 1 h. After the cornea was removed, the lens, retina and vitreous, and posterior eyecups of the RPE/choroid/sclera were dissected. The eyecups were stained with Alexa Fluor 568-conjugated isolectin B4 (Invitrogen, Carlsbad, CA, Cat # I21412) overnight at 4 °C to label invading choroidal vessels. The eyecups were then flattened by cutting radial incisions and flatmounted on a microscope slide with Fluoromount G (Southern Biotech, Birmingham, AL) for confocal imaging. The flatmounts were imaged by taking optical Z-sections at3 µm increments with a confocal microscope (FV1000, Olympus, Melville, NY). The volume (µm³) of each lesion of CNV was measured by computerized image analysis using software provided with the microscope. Lesions with obvious hemorrhage or bridging CNV were excluded. Each CNV spot was considered an independent data point. For each condition, at least 30 spots were analyzed from eight mice.

Wang H., Han X., Wittchen E.S, & Hartnett M.E. (2016). TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation. Molecular Vision, 22, 116-128.

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Quantification of Retinal Vascular Lesions

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Freshly enucleated eyes were first fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 h and followed by an additional 1 h after removing the cornea, lens, vitreous, and retina. The posterior eyecups were then blocked in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.5% Triton X-100 at room temperature, and followed by staining with AlexaFluor 568-conjugated Isolectin B4 (1:500, Invitrogen, Carlsbad, CA, USA), and αSMA (1:200, Cell Signaling Technology) overnight at 4 °C. To label αSMA staining, the eyecups were stained with FITC-conjugated goat anti-rabbit secondary antibody (1:200, Invitrogen) for 1 h at room temperature. After three washes, the eyecups were flattened by cutting incisions radially and mounted onto a microscope slide with a VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA).
Confocal images of each lectin or αSMA-labeled lesion with the z-stack set at 1 nm intervals were captured at 488 and 568 nm using a 20× objective on a Confocal Laser Scanning Microscope (Olympus Corporation, Japan). Images were imported into IMARIS (Oxford Instruments, Switzerland), and lesion volumes were determined using the Surfaces Module (Version 9.1.2, Bitplane, Santa Barbara, CA, USA). Lesions with obvious hemorrhage or bridging CNV were excluded.

Wang H., Ramshekar A., Cung T., Wallace-Carrete C., Zaugg C., Nguyen J., Stoddard G.J, & Hartnett M.E. (2023). 7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling. International Journal of Molecular Sciences, 24(12), 10276.

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Immunofluorescence Imaging of Neovessels and Arterioles

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For immunofluorescence, the section were blocked with 10% donkey serum for 60 min at room temperature, incubated with primary antibodies overnight at 4 °C and re-incubated with the corresponding secondary antibodies (1:500, Alexa 555; Invitrogen) except Alexa Fluor™ 568-conjugated isolectin B4 (IB4) (Detailed information of antibodies see Supplementary Table 1). The sections were then mounted using Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). The following antibodies were used: IB4 and α-smooth muscle actin (α-SMA). The sections were observed under a confocal laser microscope. IB4-positive neovessels and α-SMA-positive arterioles were counted using Zeiss Zen version 2.3 (Zeiss) by a double-blinded investigator. For each measurement in each mouse, we analysed 6 randomly selected regions per section (i.e. n = 6 measurements per mouse).

Guo R., Wan F., Morimatsu M., Xu Q., Feng T., Yang H., Gong Y., Ma S., Chang Y., Zhang S., Jiang Y., Wang H., Chang D., Zhang H., Ling Y, & Lan F. (2021). Cell sheet formation enhances the therapeutic effects of human umbilical cord mesenchymal stem cells on myocardial infarction as a bioactive material. Bioactive Materials, 6(9), 2999-3012.

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Quantification of Choroidal Neovascularization

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After enucleation, eyes were first fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 1 hour. After removing the cornea, lens, vitreous and retina, the posterior eyecups consisting of the RPE/choroid/sclera were fixed in 4% paraformaldehyde for 1 additional hour. After 30 minutes blocking in phosphate-buffered saline containing 1% bovine serum albumin (BSA) and 0.5% TritonX-100 at room temperature, the posterior eyecups were stained with AlexaFluor 568-conjugated Isolectin B4 (1:500, Invitrogen, Carlsbad, CA) overnight at 4°C to label invading CECs and vessels. After staining, the eyecups were flattened by cutting radial incisions and flatmounted onto a microscope slide with vectashield mounting medium (Vector Laboratories, Burlingame, CA). Confocal z-stack images of each lesion at 568 nm were acquired using a 20X objective using Confocal Laser Scanning Microscope (Olympus Corporation, Japan). Images were imported into IMARIS (the Oxford Instruments, Switzerland), and lesion volume was measured using the Surfaces Module (Version 9.1.2, Bitplane, Santa Barbara, California, USA). Laser spots with obvious hemorrhage or bridging CNV were excluded.

Wang H., Ramshekar A., Kunz E., Sacks D.B, & Hartnett M.E. (2020). IQGAP1 Causes Choroidal Neovascularization by Sustaining VEGFR2-mediated Rac1 Activation. Angiogenesis, 23(4), 685-698.

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