Unc5b

Manufactured by Cell Signaling Technology

Sourced in United States

Unc5B is a cell surface receptor that belongs to the Unc5 family. It functions as a receptor for the Netrin guidance cue, playing a role in axon guidance and cell migration.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Fluorescent mounting medium, by Agilent Technologies (1 mentions) Pdgfrβ, by Cell Signaling Technology (1 mentions) Sox17, by R&D Systems (1 mentions) Caveolin 1, by Cell Signaling Technology (1 mentions) Plvap, by BD (1 mentions) Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions) Alexa fluor 488 and 555, by Thermo Fisher Scientific (1 mentions) Lsm 510 meta, by Zeiss (1 mentions) Vascular endothelial growth factor (vegf), by Abcam (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Ab71916, by Abcam (1 mentions) Dmd108 microscope, by Leica camera (1 mentions) Halo software, by Indica Labs (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Ventana discovery xt, by Roche (1 mentions) Polypropylene difluoride membrane, by Merck Group (1 mentions) Protease inhibitor cocktail, by Boster Bio (1 mentions) Ripa buffer, by Boster Bio (1 mentions) Gapdh, by Proteintech (1 mentions)

4 protocols using unc5b

Immunolabeling of Retinal Vasculature

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Retinas were collected and placed in 3.7% formaldehyde at room temperature for 20minutes. Retinas were washed 3 × 10minutes with PBS and retinas were dissected and incubated with specific antibodies in blocking buffer (1% fetal bovine serum, 3% BSA, 0.5% Triton X-100, 0.01% Na deoxycholate, 0.02% Sodium Azide in PBS at pH 7.4) overnight at 4degree. The next day, retinas were washed 3 × 10minutes and incubated with secondary antibody for 2h at room temperature, then washed 3 × 10minute and post-fixed with 0.1% PFA for 10minutes and mounted using fluorescent mounting medium (DAKO, USA).
For mouse injected with fluorescent tracers, retinas were dissected in 3.7% formaldehyde to ensure dye fixation within the tissue during retina dissection.
The following antibodies were used: Unc5B (Cell signaling, 13851S), Claudin-5-GFP (Invitrogen, 352588), PLVAP (BD biosciences, 550563), GFAP (Millipore, MAB360), PDGFRβ (Cell Signaling, 3169S), CD13 (BD Biosciences, 558744), LEF1 (Cell Signaling, 2230S), SOX17 (R&D, AF1942), DLL4 (R&D, AF1389), Caveolin1 (Cell Signaling, D46G3), ERG (Abcam, ab92513), DAPI (Thermo Fischer, 62248). IB4 as well as all corresponding secondary antibodies were purchased from Invitrogen as donkey anti-primary antibody coupled with either Alexa Fluor 488, 568 or 647.

Furtado J., Geraldo L.H., Leser F.S., Poulet M., Park H., Pibouin-Fragner L., Eichmann A, & Boyé K. (2023). Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity. bioRxiv.

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Immunofluorescence Analysis of Angiogenic Factors

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WJ-MSC or HUVEC monolayers were grown on Lab-Tek® chamber slides with cover (Nunc, Naperville, IL, USA) up to 80% confluence, then rinsed in Hanks’ solution and fixed in 4% paraformaldehyde (15 min). Fixed cells were rinsed with Hanks’ solution, permeabilized with 0.1% Triton X-100 (20 min), and blocked (1 h) with 1% bovine serum albumin (BSA). Monolayers were incubated for 30 min at 37 °C with combinations of the following antibodies: Netrin-1 and −4 (R&D Systems), Neogenin-1 and eNOS (Santa Cruz Biotechnology, Dallas, TX, USA), UNC5b (Cell Signaling, Danvers, MA, USA), CD29 (Thermo Fisher Scientific), VEGF (Abcam, Cambridge, MA, USA) or CD31 (Sigma-Aldrich) followed by incubation with the corresponding secondary antibodies (Alexa Fluor 488 and 555; Molecular Probes, Eugene, OR, USA). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Fluorescent secondary antibodies were visualized with Zeiss LSM 510 META (Carl Zeiss Microscopy GmbH., Jena, Germany).

Prieto C.P., Ortiz M.C., Villanueva A., Villarroel C., Edwards S.S., Elliott M., Lattus J., Aedo S., Meza D., Lois P, & Palma V. (2017). Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC). Stem Cell Research & Therapy, 8, 43.

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Immunohistochemical Analysis of Endometrial Tumors

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Cre:ER^+/−Pten f/f mice were euthanized by cervical dislocation after 3 weeks of NP137 treatment. Endometrial samples were collected and formalin fixed overnight at 4 °C. Tumours were paraffin embedded for further histologic analysis. Paraffin blocks were sectioned at 3 µm and dried for 1 h at 65 °C before the pretreatment procedures of deparaffinization, rehydration and epitope retrieval in the pretreatment module at 95 °C for 20 min in 50× Tris/EDTA buffer. Before staining of sections, endogenous peroxidase was blocked. Representative images were taken with a Leica DMD108 microscope.
Immunohistochemistry was performed on an automated immunostainer (Ventana discoveryXT, Roche) using the rabbit Omni map DAB Kit. Sections were incubated with specific antibodies targeting EpCAM (no. ab71916, abcam), cleaved caspase-3 (no. 9661, Cell Signaling Technologies), netrin-1 (no. CPA2389, Cohesion Biosciences), Unc5B (no. 13851S, Cell Signaling Technologies) and CD8 (no. 4SM15, eBioscience). Staining was by anti-rabbit horseradish peroxidase, visualized with 3,3′-diaminobenzidine as a chromogenic substrate and counterstained with Gill’s haematoxylin. Histological quantifications were performed with Halo software (Indica Labs).

Cassier P.A., Navaridas R., Bellina M., Rama N., Ducarouge B., Hernandez-Vargas H., Delord J.P., Lengrand J., Paradisi A., Fattet L., Garin G., Gheit H., Dalban C., Pastushenko I., Neves D., Jelin R., Gadot N., Braissand N., Léon S., Degletagne C., Matias-Guiu X., Devouassoux-Shisheboran M., Mery-Lamarche E., Allard J., Zindy E., Decaestecker C., Salmon I., Perol D., Dolcet X., Ray-Coquard I., Blanpain C., Bernet A, & Mehlen P. (2023). Netrin-1 blockade inhibits tumour growth and EMT features in endometrial cancer. Nature, 620(7973), 409-416.

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Western Blot Protein Analysis

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Total protein was lysed in RIPA buffer (Boster, China) containing a protease inhibitor cocktail (Boster, China). Protein lysates were separated by 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis then transferred onto polypropylene difluoride membranes (Millipore, USA). The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and incubated with the following antibodies: UNC5B (1 : 1000; Cell Signaling Technology, USA), P53 (1 : 1000; R&D, USA), P21 (1 : 500; Santa Cruz, USA), and GAPDH (1 : 5000; Proteintech, China) at 4°C overnight. After washing 3 times in 1X TBST (Tris-buffered saline containing 0.1% Tween 20), the membranes were incubated with respective secondary antibodies conjugated with horseradish peroxidase (1 : 5000; Promotor, China) at room temperature for 1 hour. Eventually, the membranes were rewashed three times with TBST and visualized using an enhanced chemiluminescence kit (Beyotime, China).

Yang Z., Li H., Luo P., Yan D., Yang N., Zhang Y., Huang Y., Liu Y., Zhang L., Yan J, & Zhang C. (2021). UNC5B Promotes Vascular Endothelial Cell Senescence via the ROS-Mediated P53 Pathway. Oxidative Medicine and Cellular Longevity, 2021, 5546711.

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