Isolectin gs ib4 alexa fluor 594 conjugate

Manufactured by Thermo Fisher Scientific

Sourced in United States

Isolectin GS-IB4 Alexa Fluor 594 conjugate is a fluorescently labeled lectin derived from the seeds of the plant Griffonia simplicifolia. It selectively binds to terminal alpha-D-galactosyl residues on cell surfaces. The Alexa Fluor 594 dye provides a red fluorescent signal that can be detected using appropriate fluorescence microscopy or flow cytometry equipment.

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Lab products found in correlation

Eclipse 90i, by Nikon (2 mentions) Rabbit anti tnf α, by Abcam (1 mentions) Mouse anti mhc 2, by Abcam (1 mentions) Rabbit anti tmem119, by Abcam (1 mentions) Rabbit anti cd68, by Abcam (1 mentions) Rat anti p2ry12, by BioLegend (1 mentions) Rabbit anti ki67, by Abcam (1 mentions) Rabbit anti iba1, by Fujifilm (1 mentions) Trpa1 specific antibody, by Alomone (1 mentions) Alexa fluor 647 tagged goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions) Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Eclipse ti fluorescence microscope, by Nikon (1 mentions) Alexa fluor 594 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions) Nb300 978, by Novus Biologicals (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) Confocal laser scanning microscope, by Zeiss (1 mentions) Af 493 na, by R&D Systems (1 mentions) Rompun, by Bayer (1 mentions) Zoletil 50, by Virbac (1 mentions)

9 protocols using isolectin gs ib4 alexa fluor 594 conjugate

Immunohistochemical Analysis of Microglia in Mouse Brain

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Brains from indicated littermates were fixed using 4% paraformaldehyde (PFA) at 4°C overnight with gentle agitation, cryopreserved in 30% sucrose, frozen, and finally stored at −20°C until use. For staining, 20-μm cryosections were made and incubated in blocking/permeabilization solution containing 3% normal goat serum (NGS) and 0.2% Triton-X in PBS. The sections were treated overnight with appropriate primary antibodies diluted in 1% NGS/0.2% Triton X-100/PBS followed by the corresponding secondary antibodies for 2 hours at room temperature. The following primary antibodies were used: rabbit anti-CD68 (Abcam); mouse anti-MHC-II (Abcam); rabbit anti-Ki67 (Abcam); isolectin GS-IB4, Alexa Fluor 594 conjugate (Invitrogen); rabbit anti-TNFα (Abcam); rabbit anti-Iba1 (Wako Chemicals USA Inc.); rabbit anti-Tmem119 (Abcam); and rat anti-P2RY12 (BioLegend).

Chen H.R., Sun Y.Y., Chen C.W., Kuo Y.M., Kuan I.S., Tiger Li Z.R., Short-Miller J.C., Smucker M.R, & Kuan C.Y. (2020). Fate mapping via CCR2-CreER mice reveals monocyte-to-microglia transition in development and neonatal stroke. Science Advances, 6(35), eabb2119.

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Immunofluorescence Staining of TRPA1 in Mouse Tissues

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Glass slides with sections (4 μm) of formalin-fixed, paraffin-embedded SMA, aorta and dorsal root ganglia (DRG; positive control) of WT mice were stained with antibodies for immunofluorescence. For immunofluorescence, slides with either cross sections of blood vessels or DRG were stained with a rabbit polyclonal TRPA1-specific antibody (1:200; Alomone Labs, Israel; Cat. #: ACC-037). The fluorophore used was Alexa Fluor 647 tagged goat anti-rabbit secondary antibody (1:400 dilution; Invitrogen; Cat. #: 21244) with or without a TRPA1 blocking peptide (see Conklin et al., 2019 ). To stain cell nuclei, sections were coated with DAPI containing Slow Fade® Gold anti-fade reagent (Invitrogen; Cat. #: S36938). Fluorescence imaging was done using a Nikon eclipse Ti fluorescence microscope using NIS-Elements (Nikon; Japan) at 200X. Two filters were used for TRPA1 (Cy5 Red filter) and nuclear (DAPI filter) staining, respectively (Conklin et al., 2019 ; Jin et al., 2019a (link)). To specifically label endothelial cells, blood vessel sections were co-stained with isolectin GS-IB4 Alexa Fluor 594 conjugate (1:200 dilution; Invitrogen; Cat. #: I21413) – an endothelial cell-specific marker -- and with TRPA1 antibody (as described).

Jin L., Jagatheesan G., Lynch J., Guo L, & Conklin D.J. (2020). Crotonaldehyde-induced Vascular Relaxation and Toxicity: Role of Endothelium and Transient Receptor Potential Ankyrin-1 (TRPA1). Toxicology and applied pharmacology, 398, 115012.

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Immunostaining of GLP-1 Receptor and Intima

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We performed immunostaining using anti-GLP-1 receptor antibody (NLS1205; NOVUS Biologicals, Minneapolis, MN, USA). The sections in citrate buffer solution were heated for 15 min at 90°C in a microwave oven for antigen retrieval. After washing in phosphatebuffered saline with Tween-20 (PBST), sections were blocked with 5% donkey serum for 60 min. And then, the sections were incubated with anti-GLP-1 receptor antibody alone or with anti-α-smooth muscle actin antibody (NB300-978; NOVUS) at 25°C for 14 h. After rinsing with PBST, second antibody (Alexa Fluor 488 donkey anti-rabbit IgG only or with Alexa Fluor 594 donkey anti-goat IgG; Invitrogen Corporation, Carlsbad, CA, USA) was added to sections. To perform immunostaining of the intima, isolectin GS-IB4 Alexa Fluor 594 conjugate (Invitrogen Corporation) was added to the artery sections. After rinsing with PBST, 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was added for 5 min at 25°C. TCF7L2 staining was performed using a previously reported method. And according to the previously established method, 12

Kimura T., Obata A., Shimoda M., Okauchi S., Hirukawa H., Kohara K., Kinoshita T., Nogami Y., Nakanishi S., Mune T., Kaku K, & Kaneto H. (2017). Decreased glucagon-like peptide 1 receptor expression in endothelial and smooth muscle cells in diabetic db/db mice: TCF7L2 is a possible regulator of the vascular glucagon-like peptide 1 receptor. Diabetes & vascular disease research, 14(6).

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Retinal Whole-Mount Immunostaining

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Eyes were removed and fixed in 2% PFA for 5 min. Retinae were dissected in 2X PBS, flattened by radial incisions, and stored in −20°C methanol until staining. Retinal whole-mounts (n = 3 biological replicates from each treatment, αFzd4 or αKLH) were blocked for 1 hr with 10% serum, 0.5% Triton-X, and 0.5% Tween-20 in PBS, incubated with Isolectin GS-IB₄ Alexa Fluor 594 Conjugate (1:100, Life Technologies I21413), transferred to slides, and coverslipped with Dako fluorescence mounting medium.

Bassett E.A., Tokarew N., Allemano E.A., Mazerolle C., Morin K., Mears A.J., McNeill B., Ringuette R., Campbell C., Smiley S., Pokrajac N.T., Dubuc A.M., Ramaswamy V., Northcott P.A., Remke M., Monnier P.P., Potter D., Paes K., Kirkpatrick L.L., Coker K.J., Rice D.S., Perez-Iratxeta C., Taylor M.D, & Wallace V.A. (2016). Norrin/Frizzled4 signalling in the preneoplastic niche blocks medulloblastoma initiation. eLife, 5, e16764.

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Immunofluorescence Analysis of αGal and Neu5Gc

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Tissues were fixed using 4% paraformaldehyde and made into paraffin blocks. Tissue sections were used for the immunofluorescence analysis of two antigens. αGal was stained with isolectin GS-IB4, Alexa Fluor® 594 conjugate (Life Technologies, Carlsbad, CA, USA). Neu5Gc was stained with chicken anti-Neu5Gc antibody (BioLegend, San Diego, CA, USA) and goat anti-chicken fluorescein isothiocyanate (FITC) (Abcam, Cambridge, UK) following the manufacturer’s protocols. The tissues were imaged using a confocal laser scanning microscope (Zeiss, Oberkochen, Germany).

Shim J., Ko N., Kim H.J., Lee Y., Lee J.W., Jin D.I., Kim H, & Choi K. (2021). Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs. Transgenic Research, 30(5), 619-634.

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Intravitreal Injection Reduces Retinal Neovascularization

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Newborn mice were exposed to hyperoxia (75 ± 0.5% O₂) from postnatal day 7 to 12 and returned to normal room air. At postnatal day 14, we intravitreally injected 1 μL of PBS, KAI WT, rhKAI1 and KAI Mut into the right eyes of mice (n = 4). At P17, the enucleated eyes were prepared for immunofluorescent staining of whole-mount retinas with Alexa Fluor® 594 isolectin GS-IB4 conjugate (5 μg/mL; Invitrogen). The whole-mount retinas were viewed with a fluorescence microscope (Eclipse 90i; Nikon). Then, the neovascular tufts were marked and calculated using Image J (NIH). The area of neovascular tufts was normalized to the area of whole retina.

Lee J.W., Hur J., Kwon Y.W., Chae C.W., Choi J.I., Hwang I., Yun J.Y., Kang J.A., Choi Y.E., Kim Y.H., Lee S.E., Lee C., Jo D.H., Seok H., Cho B.S., Baek S.H, & Kim H.S. (2021). KAI1(CD82) is a key molecule to control angiogenesis and switch angiogenic milieu to quiescent state. Journal of Hematology & Oncology, 14, 148.

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Intravitreal Anti-VEGF Injection in OIR Mice

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C57BL/6 mice were obtained from Central Lab. Animal. OIR was induced in newborn mice as previously described [16 (link),17 (link)]. At postnatal day (P)14, we intravitreally injected 1 μL of phosphate-buffered saline (PBS) or anti-mouse VEGF164 antibody (1 μg/eye; cat. no.: AF-493-NA, R&D) into the right eye of mice using NanoFil 10 μL syringe with 35 gauge needle (WPI) after anesthesia using tiletamine plus zolazepam (Zoletil 50, Virbac; 30 mg/Kg) and xylazine (Rompun, Bayer; 10 mg/Kg). At P17, the mice were euthanized by carbon dioxide in deep anesthesia using tiletamine plus zolazepam and xylazine. Then, the enucleated eyes were prepared for immunofluorescent staining of whole mount retinas with Alexa Fluor 594 isolectin GS-IB4 conjugate (5 μg/mL; Invitrogen). The whole mount retinas were viewed with a fluorescence microscope (Eclipse 90i; Nikon). Then, the neovascular tufts were marked and the extent of them were calculated using NIS-Elements AR (v. 3.2; Nikon). The area of neovascular tufts was normalized to the area of whole retina. All animal studies were approved by the Seoul National University Institutional Animal Care and Use Committee and conducted in agreement with the ARVO statement for the use of animals in ophthalmic and vision research.

Jo D.H., Park S.W., Cho C.S., Powner M.B., Kim J.H., Fruttiger M, & Kim J.H. (2015). Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice. PLoS ONE, 10(7), e0134308.

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Imaging Inflammasome Activation in Organotypic Hippocampal Slices

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Images were collected on a Leica TCS SP8 AOBS upright confocal microscope through LAS X (v3.5.2.18963) using a 63X/1.40 HCS PL Apo objective and 1× confocal zoom. The confocal settings were as follows: pinhole 1 airy unit, scan speed 400 Hz bidirectional, format 1024 × 1024. The white‐light laser was used with FITC 488 nm, Cy3 555 nm, Texas Red 594 nm and Cy5 647 nm laser lines. Images were collected using hybrid and photon‐multiplying tube detectors with the following detection mirror settings: DAPI 410–520 nm, Cy3 565–636 nm and Cy5 657–744 nm for IL‐1β/Iba1 images; and DAPI 410–478 nm, FITC 498–584 nm and Texas Red 604–749 nm for ASC/Iba1 images. Images were collected sequentially to eliminate crosstalk between channels. For live imaging, OHSCs were LPS‐primed (1 µg/ml, 3 hr) and then medium was replaced with phenol‐red‐free, serum‐free culture medium containing Hoechst (2 µg/ml; ThermoFisher) and isolectin GS‐IB4–AlexaFluor 594 conjugate (from Griffonia simplicifolia; IB4; 5 µg/ml; ThermoFisher) and incubated for 2 hr at 37°. OHSCs were subsequently covered with 1·5 ml phenol‐red‐free, serum‐free medium to allow lens immersion, and then nigericin (10 µm) was spiked into the culture medium underneath the insert. The OHSCs were then placed into a confocal microscope chamber heated to 37° and imaged for 60–90 min.

Hoyle C., Redondo‐Castro E., Cook J., Tzeng T., Allan S.M., Brough D, & Lemarchand E. (2020). Hallmarks of NLRP3 inflammasome activation are observed in organotypic hippocampal slice culture. Immunology, 161(1), 39-52.

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Histological Analysis of Myocardial Tissue

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The rats were sacrificed at 28 days to harvest the heart. The harvested hearts were fixed with 4% (wt/v) paraformaldehyde overnight at 4 °C, and then immersed in 30% (wt/v) sucrose solution in the following 24 h at 4 °C. After embedding and frozen in the OCT, the blocks were then cryosectioned into sections at 10 μm. The slides with myocardial sections were further stained with Picro Sirius Red (ab150681, Abcam) to visualize the dense collagen fibers in the fibrotic area. Hematoxylin and eosin (H&E) staining was performed to examine the immune response and hydrogel engraftment in the myocardium.
The immunofluorescence staining was performed to identify the proliferation of CMs in the border zone of left ventricular tissue sections with ki67 (ab15580, 1:100, Abcam), cTnT (ab45932, 1:300, Abcam) and DAPI. To calculate the CM proliferation rate in the border zone, images at 10× per rat were taken in each group, the cells with double positives of ki67 and cTnT were considered proliferative CMs, and the number of rats in each group was 4. Also, the capillary in the border zone was stained with isolectin GS-IB4 Alexa Fluor 594 Conjugate (I21413, Thermo Fisher Scientific). To calculate the capillary density in the border zone, images at 10× per rat were taken in each group, and the number of rats in each group was 4.

Yang H., Qin X., Wang H., Zhao X., Liu Y., Wo H.T., Liu C., Nishiga M., Chen H., Ge J., Sayed N., Abilez O.J., Ding D., Heilshorn S.C, & Li K. (2019). An in Vivo miRNA Delivery System for Restoring Infarcted Myocardium. ACS nano, 13(9), 9880-9894.

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