Fitc isolectin b4

Manufactured by Vector Laboratories

Sourced in United States

FITC-isolectin B4 is a fluorescently labeled lectin from Griffonia simplicifolia that binds to terminal α-D-galactose residues on cell surfaces. It is commonly used as a marker for endothelial cells and microvascular structures.

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Lab products found in correlation

Immu mount vectashield mounting medium, by Vector Laboratories (3 mentions) A1 confocal microscope, by Nikon (2 mentions) α sarcomeric actin, by Merck Group (2 mentions) Biotinylated secondary antibody, by Vector Laboratories (2 mentions) Mouse anti neun, by Merck Group (2 mentions) Rabbit anti cleaved caspase 3, by Cell Signaling Technology (2 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Rabbit anti pten, by Abcam (2 mentions) Mouse anti parvalbumin, by Swant (2 mentions) Rabbit anti dsred, by Takara Bio (2 mentions) Rabbit anti gaba, by Merck Group (2 mentions) Rat anti somatostatin, by Merck Group (2 mentions) Mouse anti gaba, by Merck Group (2 mentions) Goat anti mcherry, by Antibodies-online (2 mentions) Alexa fluor conjugated streptavidin, by Vector Laboratories (2 mentions) Isolectin b4 dylight 594, by Vector Laboratories (2 mentions) Rabbit anti parvalbumin, by Swant (2 mentions) Goat anti ctgf, by Santa Cruz Biotechnology (2 mentions) Sudan black b, by Merck Group (1 mentions) In situ cell death detection kit, by Merck Group (1 mentions)

Close spelling variants of this product

Isolectin B4 (42 citations) FITC-conjugated Isolectin B4 (9 citations) FITC-conjugated Griffonia simplicifolia Isolectin B4 (4 citations) FITC-labeled Isolectin B4 (3 citations) FITC‐labeled GSL I‐isolectin B4 (2 citations)

14 protocols using fitc isolectin b4

Cardiac Immune Cell Profiling Post-Myocardial Infarction

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Mice (N = 10–12 per group) were sacrificed 30 days after AMI.
Following echocardiography, hearts were harvested as previously described [16 (link)]. Hearts were stained against: Rabbit
anti-mouse IBA1 (Novus Biologicals, CO) overnight at 4°C. After washing,
sections were incubated with secondary antibody conjugated to Alexa Fluor 488
(1:500, Invitrogen, Carlsbad, CA), then incubated with Sudan Black B (Sigma
Aldrich, St. Louis, MO) for 30 minutes and subsequently incubated with DAPI
nuclear counterstain. A similar protocol was used to prepare heart sections from
mice on baseline, day 7 and day 30 for capillary density assessment using FITC-
Isolectin B4 (FL1201, Vector Labs, Burlingame, CA). Apoptotic cells in heart
sections were identified at day 1 by terminal deoxynucleotidyl
transferase-mediated dUTP nick end-labeling (TUNEL) staining using the In-Situ
Cell Death Detection Kit and α-sarcomeric actin (1:200 dilution, Sigma
Aldrich, St. Louis, MO) according to the manufacturer’s protocol. DAPI
counterstaining was followed by a final PBS wash.10–15 adjacent
pre-infarct and remote zones per section were analyzed (1–2
sections/animal) at 40x magnification using Nikon Confocal Microscope A1 in the
University of Kentucky Confocal Microscopy facility.

Tripathi H., Al-Darraji A., Abo-Aly M., Peng H., Shokri E., Chelvarajan L., Donahue R., Levitan B.M., Gao E., Hernandez G., Morris A.J., Smyth S.S, & Abdel-Latif A. (2020). Autotaxin Inhibition Reduces Cardiac Inflammation and Mitigates Adverse Cardiac Remodeling After Myocardial Infarction. Journal of molecular and cellular cardiology, 149, 95-114.

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Immunostaining of Cryosectioned Embryos and Flat-Mount RPE/Choroid

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For cryosections, embryos were fixed in 4% paraformaldehyde (PFA), cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) overnight at 4°C, embedded in OCT compound (Tissue Tec; Sakura Fine Technical, Japan), and sectioned at 10 μm using a cryostat (HM560; Thermo Fisher Scientific, Waltham, MA). Specimens were blocked with horse serum for 1 hr at room temperature and incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hr at room temperature.
For flat-mount immunostaining of the RPE/choroid, the tissues were fixed with 4% PFA at room temperature for 30 min, and washed three times with PBS containing 0.5% Triton X-100 (PBST, Nacalai Tesque, Japan), incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hr at room temperature (Zhu et al., 2012 (link)). The primary antibodies and dilutions used were as follows: goat anti-Aldh1a1 (1:1,000; Abcam), rabbit anti-Aldh1a3 (1:1,000; Sigma), rat anti-endomucin (1:400; Millipore), mouse anti-GS (1:1,000; Millipore), FITC-isolectin B4 (1:100; Vector Laboratories), rabbit anti-ZO-1 (1:100; Invitrogen), rabbit anti-GFP (1:1,000; Abcam), rabbit anti-ERG (1:400; Abcam), rabbit anti-Pax6 (1:200; BioLegend), and rabbit anti-Sox9 (1:200; Millipore).

Goto S., Onishi A., Misaki K., Yonemura S., Sugita S., Ito H., Ohigashi Y., Ema M., Sakaguchi H., Nishida K, & Takahashi M. (2018). Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells. eLife, 7, e32358.

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Cardiac Tissue Analysis Post-Myocardial Infarction

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After final echocardiography and peripheral blood collection, hearts were harvested in diastole with saturated KCl and CdCl (100 mM) injected through the apex into the LV cavity. The LV apex was then cannulated and the heart perfused with PBS followed by 10% buffered formalin at 75 mmHg with severed inferior vena cava serving as the outlet. Hearts were then cut into 2 mm cross sectional slices and processed for paraffin embedding. Slices were cut into 4 μm sections for histology and immunofluorescent staining. LV area, LV cavity area, and infarct area were measured in Masson’s Trichrome stained sections. Images were acquired digitally and areas were measured using NIH ImageJ (v1.37). Cell turn-over was evaluated by staining against BrdU (Roche, Indianapolis, IN) and α-sarcomeric actin (Sigma, St Louis, MO) or myosin heavy chain (MHC) (Abcam, Cambridge, MA) and counting BrdU^posα-SA^pos/MHC^pos cells in both ischemic and remote regions. GFP bone marrow cell retention was assessed by anti-GFP staining in infarct border regions (Abcam). Capillary density was measured by staining against FITC- isolectin B4 (FL1201, Vector Labs, Burlingame, CA) in cardiac sections. 10–15 adjacent areas in the peri-infarct and remote zones per section were analyzed (1–2 sections/animal) at 40x magnification using Nikon Confocal Microscope A1 in the University of Kentucky Confocal Microscopy facility.

Klyachkin Y.M., Idris A., Rodell C.B., Tripathi H., Ye S., Nagareddy P., Asfour A., Gao E., Annabathula R., Ratajczak M., Burdick J.A, & Abdel-Latif A. (2018). Cathelicidin Related Antimicrobial Peptide (CRAMP) Enhances Bone Marrow Cell Retention and Attenuates Cardiac Dysfunction in a Mouse Model of Myocardial Infarction. Stem cell reviews, 14(5), 702-714.

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Immunocytochemistry Analysis of VEGF-A Effects

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For immunocytochemistry, HUVECs were grown to confluence on glass coverslips coated with 2% gelatin and treated with or without VEGF-A (10 ng/ml) for 30 min, followed by incubation with FITC-RLYE (100 ng/ml). Cells were fixed in 3.7% formaldehyde for 30 min and permeabilized with 0.2% Triton X-100 in PBS, then incubated with blocking solution of PBS containing 3% normal goat serum and 0.05% Tween-20. Cells were labeled with antibodies against human VE-cadherin (Santa Cruz) and human CD31 (PECAM-1, Santa Cruz). Tumor sections (30 μm) were stained as previously described [45 (link)] by incubating with one of the following antibodies: rat anti-CD31, (Pharmingen), goat anti-VE-cadherin (Santa Cruze), and rabbit anti-NG2 (Millipore) for 2 h at room temperature, rinsed in PBS, and incubated with Alexa Fluor-, FITC- or TRITC-conjugated secondary antibody for 60 or 90 min at room temperature. Some tumor sections were also incubated with FITC-isolectin B4 (5 μg/ml; Vector Laboratories, Burlingame, CA) for 1 h. The sections were mounted with Permount solution after washing three times with PBS. Images were photographed and analyzed using a confocal fluorescence microscope. Nuclear staining was performed with 4’, 6-diamidino-2-phenylindole (DAPI, 1 ng/ml), and apoptotic cells were detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (Roche, Korea).

Baek Y.Y., Lee D.K., Kim J., Kim J.H., Park W., Kim T., Han S., Jeoung D., You J.C., Lee H., Won M.H., Ha K.S., Kwon Y.G, & Kim Y.M. (2016). Arg-Leu-Tyr-Glu tetrapeptide inhibits tumor progression by suppressing angiogenesis and vascular permeability via VEGF receptor-2 antagonism. Oncotarget, 8(7), 11763-11777.

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Quantifying Choroidal Neovascularization in Mice

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One to four weeks after the CNV Laser procedure, mice were euthanized by cervical dislocation and eyes were enucleated and fixed at 4% paraformaldehyde solution for 1–2 h. Flat mounts of RPE-choroid complex were obtained by removing the anterior segments and the neural retina under an operation microscope. Then retinal and choroid tissues were washed in PBS containing 1% BSA, 0.2% Tween-20 (AppliChem GmbH, Darmstadt 64291, Germany) and 0.1% Triton-X 100. Eyecups were incubated with 0.5% fluorescein-isothiocyanate (FITC)-isolectin B4 (Vector Laboratories Inc., Burlingame, CA 94010, USA) overnight at 4 °C to label invading choroid vessels and then washed in PBS three times. After staining, the eyecups were flattened by four to six radial cuts from the edge to the equator and flat mounted with the scleral side facing down onto a microscope slide. The flatmounts were analyzed with fluorescence microscope (Olympus, Hamburg, Germany). The size of FITC-isolectin B4 positive CNV areas was quantified with Olympus CellF software. Lesions with obvious hemorrhage or bridging CNV were excluded. Images were measured and confirmed by two reviewers in a masked procedure.

Wang X., Ma W., Han S., Meng Z., Zhao L., Yin Y., Wang Y, & Li J. (2017). TGF-β participates choroid neovascularization through Smad2/3-VEGF/TNF-α signaling in mice with Laser-induced wet age-related macular degeneration. Scientific Reports, 7, 9672.

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Retinal Vascular Morphometry in CMG2 Knockout Mice

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Progeny of a het-het cross of CMG2-ΔTM-ICD were collected and euthanized by decapitation. Eyes were dissected from the head using scalper and forceps and fixed in 4% PFA for 30–120 min., then transferred to PBS. Retinas were dissected by removing the cornea and iris, then using forceps to dissect the choroid away from the retina and lens. The lens and then hyaloid vessels were then removed and the retina permeabilized and blocked overnight in 0.5% Triton X-100 and 1% BSA in PBS at 4°C. Retinas were stained with 1:200 FITC-Isolectin B4 (Vector Labs, Burlingame, CA, USA) in PBS containing 1mM CaCl2 for 24–72 hours. After staining, retinas were washed 5× 10 min. in PBS at room temperature. Retinas were then relaxed using four radial cuts and they were mounted under coverslips using SlowFade Gold antifade (ThermoFischer, Burlington, MA, USA). Retinas of CMG2^wt and CMG2^−/− animals were then imaged under standard illumination and exposure with a Zeiss Axiophot Upright Florescence Microscope in tiling mode (4×4 mosaic from the center of optic nerve) at 5x magnification and measured by an investigator blinded to genotype. Arterial branching was scored using the rubrik in Supplemental File 1.

Cryan L.M., Tsang T.M., Stiles J., Bazinet L., Lee S.L., Garrard S., Madrian E., Roberts C., Payne J., Jensen A., Frankel A.E., Ackroyd P.C., Christensen K.A, & Rogers M.S. (2022). Capillary Morphogenesis gene 2 (CMG2) mediates growth factor-induced angiogenesis by regulating endothelial cell chemotaxis. Angiogenesis, 25(3), 397-410.

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Quantifying Laser-Induced Choroidal Neovascularization

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Fourteen days after laser injury, eyes were enucleated and fixed with 4% paraformaldehyde (PFA). The eye cups were blocked for 30 min and then incubated overnight at 4 °C with 0.5% FITC-isolectin B4 (Vector Laboratories, Burlingame, CA). Then, the RPE-choroid-sclera complex was flatmounted in an antifade medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories). Flatmounts were examined with a confocal microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were visualized at a wavelength of 488 nm, and the capture emission was between 515 to 545 nm. Horizontal optical sections (1 μm steps) were obtained from the surface of the RPE-choroid-sclera complex. The volume of CNV fluorescence was measured with software from the microscope manufacturer (TCS SP; Leica).

Zhuang Z., qin X., Hu H., Tian S.Y., Lu Z.J., Zhang T.Z, & Bai Y.L. (2015). Down-regulation of microRNA-155 attenuates retinal neovascularization via the PI3K/Akt pathway. Molecular Vision, 21, 1173-1184.

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Visualizing Breast Cancer Metastasis

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Control shRNA or Nck-depleted (shNck) MDA-MB-231 breast carcinoma cells were fluorescently labeled with CellTracker Orange CMRA dye (Molecular Probes, Eugene, OR) before injection (5 × 10⁵ cells) into the tail vein of 8-wk-old female nude mice (n = 5/treatment). Animals were killed 36 h after receiving cell injections, and lungs were collected for histological processing. Paraffin embedded sections (4 µm thick) spaced every 50 µm of lung tissue were subjected to staining with fluorescein isothiocyanate (FITC)-Isolectin-B4 (Vector Laboratories, Burlingame, CA), en endothelial cell marker for visualization of the lung vasculature. At least five tissue sections/lung were examined using a Zeiss LSM780 confocal microscope equipped with a Plan-Apo 20X/0.8NA objective.

Morris D.C., Popp J.L., Tang L.K., Gibbs H.C., Schmitt E., Chaki S.P., Bywaters B.C., Yeh A.T., Porter W.W., Burghardt R.C., Barhoumi R, & Rivera G.M. (2017). Nck deficiency is associated with delayed breast carcinoma progression and reduced metastasis. Molecular Biology of the Cell, 28(24), 3500-3516.

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Visualizing and Quantifying Choroidal Neovascularization in Mice

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After laser injury, mice were euthanized, and eyes were enucleated and fixed with 4% paraformaldehyde for 30 minutes at 4°C. Eyecups were incubated with 0.7% fluorescein isothiocyanate (FITC)-isolectin B4 (Vector Laboratories, Burlingame, CA, USA), and R-phycoerythrin-conjugated anti-F4/80 (Bio-Rad, Hercules, CA, USA) and the flat mounts of RPE-choroid-sclera were mounted in antifade medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories). CNV volume was visualized using a scanning laser confocal microscope (Nikon A1R, Nikon Instruments). Volumes were quantified using Image J software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) as previously reported.⁵³ (link) F4/80 labeling was quantified by densitometry of the F4/80 signal in the maximum z-projection of the CNV lesion.

Makin R.D., Argyle D., Hirahara S., Nagasaka Y., Zhang M., Yan Z., Kerur N., Ambati J, & Gelfand B.D. (2020). Voluntary Exercise Suppresses Choroidal Neovascularization in Mice. Investigative Ophthalmology & Visual Science, 61(5), 52.

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Quantifying Choroidal Neovascularization in RPE

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At 1 week after the laser injury, eyes were enucleated and fixed with 4% paraformaldehyde for 30 minutes at 4°C. RPE flat mounts were obtained after the neurosensory retina was gently detached and severed from the optic nerve. The flat mounts were then washed in PBS, followed by dehydration and rehydration through a methanol series. After blocking in PBS with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 0.5% Triton X-100 (Sigma-Aldrich) for 1 hour at room temperature, flat mounts were incubated with 0.7% FITC–isolectin B4 (Vector Laboratories, Burlingame, CA, USA) overnight at 4°C. The RPE–choroid–sclera complex was then flat mounted in antifade medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories) and cover slipped. Choroidal neovascularization was visualized using a blue argon laser wavelength (488 nm) and a scanning laser confocal microscope (A1R confocal microscope system; Nikon, Tokyo, Japan), and quantified using FIJI software (http://fiji.sc/).

Narendran S., Pereira F., Yerramothu P., Apicella I., Wang S.B., Varshney A., Baker K.L., Marion K.M., Ambati M., Ambati V.L., Ambati K., Sadda S.R., Gelfand B.D, & Ambati J. (2020). A Clinical Metabolite of Azidothymidine Inhibits Experimental Choroidal Neovascularization and Retinal Pigmented Epithelium Degeneration. Investigative Ophthalmology & Visual Science, 61(10), 4.

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