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Biotin conjugated isolectinb4 ib4

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Biotin-conjugated isolectinB4 (IB4) is a fluorescently labeled lectin used as a tool for histochemical and cytochemical applications. It binds specifically to terminal alpha-D-galactose and N-acetyl-alpha-D-galactosamine residues on cell surfaces. This product can be used to identify and study various cell types in biological samples.

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Lab products found in correlation

Alexa fluor 488 conjugated streptavidin, by Thermo Fisher Scientific (3 mentions) Protein block serum free, by Agilent Technologies (1 mentions) Rabbit anti collagen 4, by Bio-Rad (1 mentions) Alexa fluor 647 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Eclipse ti inverted epifluorescence microscope, by Nikon (1 mentions) Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Anti neun, by Merck Group (1 mentions) Murine tnf α elisa development kit, by Thermo Fisher Scientific (1 mentions) Ru28362, by Merck Group (1 mentions) Ecl plus western blotting reagent, by Cytiva (1 mentions) 3h corticosterone, by Cytiva (1 mentions) Immobilon p membrane, by Merck Group (1 mentions)

6 protocols using biotin conjugated isolectinb4 ib4

1

Quantifying Retinal Angiogenesis in Postnatal Mice

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Eyes from postnatal day 5 and day 6 pups mice were fixed in 4% paraformaldehyde for 30 min. Retinas were dissected and permeabilized overnight in PBS containing 1% BSA and 0.5% Triton X-100. The permeabilized retinas were incubated with biotin-conjugated isolectinB4 (IB4) (20μg/ml, Sigma-Aldrich), followed by Alexa Fluor 488–conjugated streptavidin (Invitrogen Life Technologies) co-stained with or without ERG (EC marker) antibody, and whole mounts were photographed under a fluorescence microscope. The total length and number of branch points of IB4-positive vessels in the entire retina were quantified on composite high-magnification images using Image J software (v 1 .52). In vivo proliferation of ECs in retina was measured by the BrdU assay following intraperitoneal injection of 300 μg of BrdU (Sigma #B5002) per P6 pup that weights about 3gm and then co-stained with anti-BrdU antibody and IB4 antibody (n=6 per group), as reported 57 (link), 58 (link).
Das A., Ash D., Fouda A.Y., Sudhahar V., Kim Y.M., Hou Y., Hudson F.Z., Stansfield B.K., Caldwell R.B., McMenamin M., Littlejohn R., Su H., Regan M.R., Merrill B.J., Poole L.B., Kaplan J.H., Fukai T, & Ushio-Fukai M. (2022). Cysteine Oxidation of Copper transporter SLC31A1/CTR1, drives VEGFR2 signaling and Angiogenesis. Nature cell biology, 24(1), 35-50.
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2

Retina and Cornea Whole-Mount Staining

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Retinas were dissected and fixed as described previously (46 (link)) and then whole-mount stained with rabbit anti–collagen IV (Bio-Rad, 2150-1470), Alexa Fluor 488–conjugated anti-rabbit IgG (Thermo Fisher Scientific, A11008) or biotin-conjugated isolectin B4 (IB4) (Sigma-Aldrich, L2140), and Alexa Fluor 647–conjugated streptavidin (Thermo Fisher Scientific, S21374), which were all diluted in PBS containing 10% serum-free protein block (Dako) and 0.1% Tween-20. Dissected corneas were fixed in 2% formaldehyde for 1 hour and then whole-mount stained with Cy3-conjugated neuron-specific beta-III tubulin (R&D Systems, NL1195R) or FITC-conjugated rat anti–mouse PECAM-1 (CD31) (BD Biosciences, 558738). Antibodies were diluted in PBS containing 3% BSA and 0.1% Triton X-100. Staining was visualized with an Eclipse Ti inverted epifluorescence microscope (Nikon).
Brash J.T., Denti L., Ruhrberg C, & Bucher F. (2019). VEGF188 promotes corneal reinnervation after injury. JCI Insight, 4(21), e130979.
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3

Retinal Angiogenesis Quantification

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Eyes from postnatal day 5 (p5) mice were enucleated and fixed in 4% paraformaldehyde for 30 min. Retinas were dissected and permeabilized overnight in PBS containing 1% BSA and 0.5% Triton X-100. The permeabilized retinas were incubated with biotin-conjugated isolectinB4 (IB4) (20 μg/ml, Sigma-Aldrich), followed by Alexa Fluor 488-conjugated streptavidin (Invitrogen Life Technologies). After washes, samples were flat-mounted using Vectashield (Vector Labs) mounting medium and imaged under a fluorescence microscope. The total length, number of branch points and tip cells of IB4-positive vessels in the retina were quantified by two independent investigators in a blind fashion on composite high-magnification images using Image J software (v 1 0.52).
Nagarkoti S., Kim Y.M., Ash D., Das A., Vitriol E., Read T.A., Youn S.W., Sudhahar V., McMenamin M., Hou Y., Boatwright H., Caldwell R., Essex D.W., Cho J., Fukai T, & Ushio-Fukai M. (2022). Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis. Angiogenesis, 26(1), 77-96.
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4

Immunohistochemical Profiling of Neuroinflammation

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NMDA, α-AA, the mouse monoclonal anti-glial fibrillary acidic protein (GFAP), and the biotin-conjugated isolectin B4 (IB4) from Bandeiraea simplicifolia were all purchased from Sigma (St. Louis, MO). The mouse monoclonal anti-NeuN was purchased from Chemicon (Temecula, CA), the mouse anti-rat CD11b antibody (clone MRC OX-42) was from Serotec Ltd. (Oxford, UK), and the rabbit polyclonal anti-S100B antibody was from DAKO (Dako Diagnostics, Barcelona, Spain). The goat polyclonal AMCase (M-19) antibody that specifically binds the transcription factor YM1 was from Santa Cruz Biotechnology (Santa Cruz, CA) as was the rabbit polyclonal anti-GR antibody. Secondary antibodies and immunohistochemical reagents were from Sigma.
[3H]PK-11195 was purchased from Perkin-Elmer (Boston, MA). [3H]Corticosterone was from Amersham Bioscience (Bucks, UK) and RU-28362 was purchased from Sigma. Dodecyl sulphate-polyacrylamide gel electrophoresis standards were purchased from Bio-Rad (Hercules CA), Immobilon-P membranes were from Millipore (Bedford, MA), and ECL Plus Western Blotting reagent was from Amersham Bioscience (Bucks, UK). The murine TNF-α ELISA development kit was purchased from PeproTech (Paris, France).
Batlle M., Ferri L., Andrade C., Ortega F.J., Vidal-Taboada J.M., Pugliese M., Mahy N, & Rodríguez M.J. (2015). Astroglia-Microglia Cross Talk during Neurodegeneration in the Rat Hippocampus. BioMed Research International, 2015, 102419.
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5

RPE/Choroid Whole-Mount and TUNEL Assay for CNV

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To prepare RPE/choroid whole‐mounts, enucleated eyes were initially fixed in 2% PFA overnight. Eyes were dissected, and RPE/choroidal tissues were blocked and permeabilized in 5% BSA with 0.3% Triton X‐100 in PBS for 2 h. For evaluation of CNV formation, the neovascular membrane was stained with biotin‐conjugated isolectin B4 (IB4; Sigma‐Aldrich, St Louis, MO, USA; L2140; 1:100), followed by incubation with Rhodamine Red‐X‐labelled streptavidin (Jackson Immuno Research Laboratories, West Grove, PA, USA; 016‐290‐084; 1:400). The CNV volume was measured using a series of Z‐stack images (from the surface to the deepest focal plane) using Volocity® Image Analysis Software 6.0. TMR Red‐dUTP TUNEL (Roche Diagnostics, Burgess Hill, UK) staining of DNA breaks was performed on 10 µm cryosections of enucleated eyes from treated mice, according to the manufacturer's protocol.
Theodoropoulou S., Copland D.A., Liu J., Wu J., Gardner P.J., Ozaki E., Doyle S.L., Campbell M, & Dick A.D. (2016). Interleukin‐33 regulates tissue remodelling and inhibits angiogenesis in the eye. The Journal of Pathology, 241(1), 45-56.
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6

Quantifying Retinal Angiogenesis in Postnatal Mice

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Eyes from postnatal day 5 and day 6 pups mice were fixed in 4% paraformaldehyde for 30 min. Retinas were dissected and permeabilized overnight in PBS containing 1% BSA and 0.5% Triton X-100. The permeabilized retinas were incubated with biotin-conjugated isolectinB4 (IB4) (20μg/ml, Sigma-Aldrich), followed by Alexa Fluor 488–conjugated streptavidin (Invitrogen Life Technologies) co-stained with or without ERG (EC marker) antibody, and whole mounts were photographed under a fluorescence microscope. The total length and number of branch points of IB4-positive vessels in the entire retina were quantified on composite high-magnification images using Image J software (v 1 .52). In vivo proliferation of ECs in retina was measured by the BrdU assay following intraperitoneal injection of 300 μg of BrdU (Sigma #B5002) per P6 pup that weights about 3gm and then co-stained with anti-BrdU antibody and IB4 antibody (n=6 per group), as reported 57 (link), 58 (link).
Das A., Ash D., Fouda A.Y., Sudhahar V., Kim Y.M., Hou Y., Hudson F.Z., Stansfield B.K., Caldwell R.B., McMenamin M., Littlejohn R., Su H., Regan M.R., Merrill B.J., Poole L.B., Kaplan J.H., Fukai T, & Ushio-Fukai M. (2022). Cysteine Oxidation of Copper transporter SLC31A1/CTR1, drives VEGFR2 signaling and Angiogenesis. Nature cell biology, 24(1), 35-50.
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