Embryonic brains, skin, heart or lungs were dissected from pregnant females at E15.5 and E17.5 and postnatal brains from P0 and P7 pups. P22 mice were euthanized with isoflurane overdose and the brains were perfused with 4% paraformaldehyde (PFA). All other brains and organs were drop fixed in 4% PFA overnight. Immunohistochemistry was carried out essentially as described previously on 50 μm-thick Vibratome sections (Ma et al. 2012 (link), 2013 (link)). Primary antibodies or reagents used include biotinylated isolectin B4 (Vector Lab), rat anti-mouse CD31 (BD Pharmingen), rabbit anti-GFP (Life Technologies), rabbit anti-Brain lipid binding protein (Millipore), rabbit anti-Desmin (Millipore), mouse anti-NeuN (Millipore) and rat anti-mouse endomucin (Affymetrix eBioscience). FITC or Cy3 conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Images were taken under a Nikon eclipse Ti microscope and processed using Adobe Photoshop CS6.
Biotinylated isolectin b4
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated isolectin B4 is a carbohydrate-binding protein derived from the seeds of the Griffonia simplicifolia plant. It has a high affinity for terminal α-D-galactose and N-acetyl-α-D-galactosamine residues, and is commonly used as a histochemical marker for microvascular endothelial cells and neural tissue.
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Lab products found in correlation
Fluoromount g, by Southern Biotech (4 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Anti laminin, by Abcam (2 mentions)
Alexa fluor streptavidin conjugated antibodies, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Cy3 or fitc conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Rat anti mouse cd31, by BD (1 mentions)
Cyanine3 (cy3), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Carl Roth (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by AppliChem (1 mentions)
Streptavidin alexa fluor 555 conjugate, by Thermo Fisher Scientific (1 mentions)
Streptavidin alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Lsm750, by Zeiss (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
27 protocols using biotinylated isolectin b4
1
Embryonic and Postnatal Tissue Immunohistochemistry
2
Retinal Vascular Network Visualization
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Isolated eyes were fixed for 30 min in 4% paraformaldehyde (Carl Roth GmbH, Karlsruhe, Germany) dissolved in phosphate-buffered saline (PBS) and washed twice for five minutes in PBS. The eyeball was opened, and the retina separated, incised to obtain a four-leaf clover shape. For staining, the retina was incubated in blocking buffer (1% BSA, Sigma-Aldrich, Munich, Germany, #A7030, and 0.3% Triton X100, AppliChem, Darmstadt, Germany, #A4975, in PBS) for two hours at room temperature or at 4 °C overnight, washed with PBS, and incubated with biotinylated isolectin B4 (Vector Laboratories, Eching, Germany; B1205, 1:25) or the primary antibody against the endothelial cell-specific molecule 1 (ESM1) (R&D Systems, Minneapolis, USA; AF1999, 1:100) overnight at 4 °C. ESM1 is a marker particularly for tip cells in a developing vasculature. After repeated washing, the retina was incubated with streptavidin conjugated with Cy3 (SigmaAldrich, Munich, Germany; S6402, 1:200) or a secondary antibody conjugated with Alexa488 (Invitrogen Life technologies, Darmstadt, Germany; A-11055, 1:200). Stained retinas were mounted on glass slides and inspected by a fluorescent confocal microscope (Leica TCS SP8 HCS A, Leica Microsystems, Wetzlar, Germany).
3
Retinal Vascularization Assay in Mice
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All animal experiments were approved by the local ethics committee (BMWFW-66.009/0155-WF/V/3b/2015). C57Bl/6 wild type littermates were injected at postnatal days P5 and P6 with 50 μg/g 79-6 (Calbiochem – Merck, Darmstadt, Germany) in 100 μl vehicle (peanut oil + 10% ethanol) or vehicle only. Retinas were isolated at P7 and stained with biotinylated isolectin B4 (Vector Laboratories, Burlingame, CA) and streptavidin-AlexaFluor555 conjugate (Invitrogen). Whole-mount retinas were analyzed with a Zeiss LSM780 confocal microscope, Photoshop CS4 and HistoQuest 3.5 software (TissueGnostics, Vienna, Austria).
4
Multimodal Cardiac Tissue Analysis
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Frozen hearts were sectioned into 10 μm thick sections at the cryostat. After washing with PBS sections were fixed by incubating in 2% formaldehyde solution for 10 min. Primary antibodies against rabbit PCM-1 (1:100, Santa Cruz, sc-67204), mouse SMA-Cy3 (1:1000, SigmaAldrich, C6198), and biotinylated isolectin B4 (Vector labs, B-1205, 1:500) were diluted in blocking buffer (PBS, 4% donkey serum, 0.1% Triton X-100 in PBS, 2 mM EDTA) and sections were incubated overnight at RT. Sections were then washed 3x with PBS (15 min) and stained with matching secondary antibody anti-rabbit Alexa Fluor® 488 (1:500, Jackson ImmunoResearch, 711-546-152) and Streptavidin-Alexa Fluor 647® (1:1000, ThermoFisher, S21374) in PBS. Subsequently, Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 or 488 dyes (ThermoFisher, C10340 and C10637) was used to detect EdU. Slides were then washed and mounted using ProLong Gold Antifade Mountant with DAPI (ThermoFisher, P36931). Images were acquired using a Keyence BZ X800 fluorescence microscope (Keyence, Japan) and Zeiss LSM 750 confocal microscopy from at least three regions of the left ventricle per heart.
5
Cardiac and Skeletal Muscle Histology
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Hearts were fixed with 4% formalin, embedded in paraffin, cut into 4 µm thick longitudinal sections and stained with hematoxylin-eosin. 10 µm frozen skeletal muscle sections of the lower leg of the hind limb were used to stain myofibril membranes using anti-laminin (Abcam) followed by anti−rabbit-Alexa 647 (Invitrogen) and capillaries with biotinylated isolectin B4 (Vector) followed by SAV-Alexa 488 (Invitrogen).
6
Isolectin B4 Staining of Rat Eyes
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Histology and electron microscopy studies were performed as previously described15 (link), including the following modifications for isolectin B4 staining: enucleated rat eyes were fixed in 1% formalin/1.25% glutaraldehyde, as this was found to be an ideal eye fixative17 (link). After fixation for approximately 72 hours, the eyes were dehydrated and paraffin embedded. Paraffin sections were cut at 4 µm, placed on poly-L-lysine slides, and rehydrated. The slides were then subjected to antigen retrieval and lectin staining using biotinylated isolectin B4 (Vector Laboratories, Burlingame, CA, USA) at 5 µg/ml followed by standard streptavidin-HRP and 3,3′-diaminobenzidine detection. The stained sections were scanned using a NanoZoomer 2.0-HT digital slide scanner (Hamamatsu, Hamamatsu City, Japan).
7
Imaging and Quantification of Retinal Vasculature
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The retina vasculature was stained as described before [81 (link)]. In brief, retinas were equilibrated with PBLEC (1 mM MgCl2, 1 mM CaCl2, 0.1 mM MnCl2, and 1% Triton X-100 in PBS) and incubated with biotinylated isolectin-B4 (1:25, Vector Laboratories, Burlingame, CA) overnight, washed, then incubated with streptavidin Alexa Fluor-594 (1:500, Thermo Fisher Scientific, Waltham, MA) and mounted with ProLong Gold anti-fade reagent (Thermo Fisher Scientific, Waltham, MA). Images were acquired using a Leica SP5 confocal microscope (Leica Biosystems, Wetzlar, Germany). Lesions were marked as the areas of condensed vascular plexus at the leading edge of the retinal vasculature. Fiji ImageJ software was used to quantify vascular coverage of the whole retina and lesions.
8
Postnatal Mouse Retina Immunostaining
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For postnatal mouse retina immunostaining, eyes were collected and fixed in 4% PFA in PBS for 20 min at room temperature. After microdissection, retinas were fixed in 4% PFA for an additional 45 min, followed by two PBS washes of 10 min each. Retinas were blocked and permeabilized with PBTS buffer (0.3% Triton X-100, 3% FBS and 3% donkey serum) for 1 h. Samples were then incubated overnight at 4 °C in biotinylated isolectin B4 (diluted 1:50; Vector Laboratories, B-1205) and primary antibodies (Supplementary Table 2 ) diluted in PBTS buffer. After five washes of 20 min each in PBTS buffer diluted 1:2, samples were incubated for 2h at room temperature with Alexa-conjugated secondary antibodies (Thermo Fisher). After three washes of 30 min each in PBTS buffer (diluted 1:2), and two washes of 10 min each in PBS, retinas were mounted with Fluoromount-G (SouthernBiotech).
9
Immunofluorescence Staining of ECM Proteins
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Primary antibodies: Rat anti-mouse PECAM-1 MEC13.3 (1:100, BD Pharmingen), rabbit anti-Fibronectin (1:200, Hynes lab) [16 (link)], mouse anti-EIIIA fibronectin antibody (1:100, Abcam, IST-9), rabbit anti-Collagen IV (1:400, Abcam, ab19808), rabbit anti-Desmin (1:200, Abcam, ab15200), biotinylated Isolectin-B4 (1:50, Vector Labs, B-1205), mouse anti-EIIIB fibronectin (1:100, Amy McMahon, Hynes lab). Secondary antibodies were Alexa488, Alexa594, and Alexa647 conjugated antibodies (1:500, Invitrogen), and Alexa-Fluor–streptavidin-conjugated antibodies (1:200, Molecular Probes).
10
Retinal Vasculature Labeling and Quantification
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Eyes were fixed in 4% PFA at 4 °C for 2 hr and washed in PBS. Retinas were dissected, permeabilized and blocked in PBS containing 1% BSA and 0.5% Triton at 4 °C overnight, washed in PBLec (1% Triton X-100, 1 mM CaCl2, 1 mM MgCl2, and 0.1 mM MnCl2 in PBS), and incubated overnight in PBLec containing biotinylated isolectin B4 (1:25, Vector Labs). After three 20-minute washes in PBS, samples were incubated with FITC-streptavidin (1:100, Vector Labs) in PBS containing 0.5% BSA and 0.25% Triton X-100 at room temperature for 2 hr. Retinas were either flat mounted using Vectashield mounting medium (Vector Labs) or processed for labeling of mitotic cells using phospho-histone H3 S10 antibody (1:100, Cell Signaling 9701). The images have been taken in the same region of the retinas. The branching points, EC area, and mitotic cells per fields were quantified from multiple experiments as described previously (Gerhardt et al., 2003).
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