Vectashield kit

Manufactured by Vector Laboratories

Sourced in United States

The Vectashield Kit is a mounting medium designed for fluorescence microscopy applications. It is formulated to preserve fluorescent signals and prevent fading. The kit includes the Vectashield mounting medium and a coverslip.

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Lab products found in correlation

Lsm 510 meta, by Zeiss (4 mentions) Fv1200, by Olympus (2 mentions) Fv3000, by Olympus (2 mentions) Lsm 710 duo confocal microscope, by Zeiss (2 mentions) Blocking one histo, by Nacalai Tesque (1 mentions) Methanol, by Nacalai Tesque (1 mentions) Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions) Protein block serum free solution, by Agilent Technologies (1 mentions) Bodipy, by Thermo Fisher Scientific (1 mentions) Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions) Nile blue chloride, by Merck Group (1 mentions) Nile red, by Merck Group (1 mentions)

5 protocols using vectashield kit

Multicolor Staining of RAW 264.7 Cells

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Fed RAW 264.7 were fixed overnight with 4% PFA, washed with PBS, and incubated for 30 minutes at room temperature with Bodipy (1mg/ml, from Thermo Fisher Scientific), WGA (5mg/ml, from Thermo Fisher Scientific), Nile Blue Chloride (10mg/ml, from Sigma) or Nile Red (5mg/ml, from Sigma). Stained cells were rinsed with PBS and mounted using a Vectashield Kit (Vector Laboratories, Inc., Burlingame, CA).
For antibody staining, after fixation cells were washed with PBS and permeabilized with PBS/0.2%TritonX-100. After 1hr blocking with PBS/0.2%TritonX-100/3%BSA at room temperature, cells were incubated overnight at 4 C with the specific primary antibody (1:100) in blocking buffer and for 1 hr at room temperature with secondary antibodies (Alexa Fluor) at a concentration of 1:500. Cells were mounted using a Vectashield Kit (Vector Laboratories, Inc., Burlingame, CA).

Villani A., Benjaminsen J., Moritz C., Henke K., Hartmann J., Norlin N., Richter K., Schieber N.L., Franke T., Schwab Y, & Peri F. (2019). Clearance by Microglia Depends on Packaging of Phagosomes into a Unique Cellular Compartment. Developmental cell, 49(1).

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Fluorescence Microscopy Imaging Protocol

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Cells on coverslips were fixed with 4% paraformaldehyde or 100% cold methanol (Nacalai Tesque) and blocked with Blocking One Histo (Nacalai Tesque). Slides were incubated with primary antibodies and then with Alexa Fluor fluorescent-conjugated secondary antibodies. The coverslips were mounted using the Vectashield kit (Vector Laboratories, Burlingame, CA, USA). The fluorescent images were collected and merged with the microscope systems FV1200 or FV3000 equipped with a laser-scanning Fluoview apparatus and software (both from Olympus, Tokyo, Japan (accessed on 10 January 2023)). The images in the figures are representative of 3 images and were analyzed using the Image J software (https://imagej.nih.gov/ (accessed on 1 July 2023)).

Fukatsu S., Okawa M., Okabe M., Cho M., Isogai M., Yokoi T., Shirai R., Oizumi H., Yamamoto M., Ohbuchi K., Miyamoto Y, & Yamauchi J. (2024). Modulating Golgi Stress Signaling Ameliorates Cell Morphological Phenotypes Induced by CHMP2B with Frontotemporal Dementia-Associated p.Asp148Tyr. Current Issues in Molecular Biology, 46(2), 1398-1412.

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Immunofluorescence Microscopy of Cultured Cells

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Cells on coverslips were fixed with 4% paraformaldehyde (Nacalai Tesque) or 100% cold methanol (Nacalai Tesque) and blocked with Blocking One. Slides were incubated with a primary antibody followed by an Alexa-Fluor fluorescence-conjugated secondary antibody. Coverslips were mounted using the Vectashield kit (Vector Laboratories, Burlingame, CA, USA). The fluorescent images were collected and integrated on a microscope FV1200 or FV3000 system (both Olympus, Tokyo, Japan) equipped with a laser scanning Fluoview instrument and its software (Olympus). The image in Figure 1A is representative of three images and was analyzed using Image J software (https://imagej.nih.gov/, accessed on 1 May 2023).

Shirai R., Cho M., Isogai M., Fukatsu S., Okabe M., Okawa M., Miyamoto Y., Torii T, & Yamauchi J. (2023). FTD/ALS Type 7-Associated Thr104Asn Mutation of CHMP2B Blunts Neuronal Process Elongation, and Is Recovered by Knockdown of Arf4, the Golgi Stress Regulator. Neurology International, 15(3), 980-993.

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Immunofluorescence Staining of Cholangiocytes

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Polarized WT and Cftr-KO cholangiocytes were processed for immunofluorescence staining. Briefly, cells were fixed with either a) 100% methanol and washed with PBS; b) or 3.7% PFA, washed using PBS/10mM glycine, and permeabilized with PBS/0.2%TritonX-100. Unspecific binding sites were blocked with Normal Horse Serum (1 (link):20 (link)) or with 3% non-fat milk for 45 minutes at RT. Cells were incubated overnight at 4°C with specific primary antibody in blocking buffer (supplementary table 1) and for 1 hour at RT with the proper secondary antibody (conjugated with Alexa Fluor 488, 555, 594 or 647 in PBS/BSA/Gly). Cells were then mounted using a Vectashield Kit (Vector Laboratories, Inc, Burlingame, CA) with 4’,6-diamidino-2-phenylindole (DAPI). Confocal analysis was performed using a Zeiss LSM 710 Duo confocal microscope or a Zeiss LSM 510 Meta with x63, 1.4 NA objective. Serial optical sections (0.5 μm thick) were collected for 3D analysis.

Fiorotto R., Villani A., Kourtidis A., Scirpo R., Amenduni M., Geibel P.J., Cadamuro M., Spirli C., Anastasiadis P.Z, & Strazzabosco M. (2016). CFTR controls biliary epithelial inflammation and permeability by regulating Src tyrosine kinase activity. Hepatology (Baltimore, Md.), 64(6), 2118-2134.

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Immunofluorescence Staining of Cholangiocytes

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Mouse and human cholangiocytes were fixed with either a) 1:1 methanol/acetone solution and washed with PBS; b) or with 4% PFA, washed with PBS/10mM glycine, and permeabilized with PBS/TritonX-100 0.2%. Unspecific binding sites were blocked with Protein Block Serum-Free solution (Dako, Agilent Technologies, Santa Clara, CA) for 1 hour at RT. Cells were then incubated overnight at 4°C with specific primary antibody (see supplementary table 1) and with the proper secondary antibody for 1 hour at RT. Cells were then mounted using a Vectashield Kit (Vector Laboratories, Burlingame, CA) with 4’,6-diamidino-2-phenylindole (DAPI). Confocal analysis was performed using a Zeiss LSM 710 Duo confocal microscope or a Zeiss LSM510 Meta.

Fiorotto R., Amenduni M., Mariotti V., Fabris L., Spirli C, & Strazzabosco M. (2018). Src kinase inhibition reduces inflammatory and cytoskeletal changes in ΔF508 human cholangiocytes and improves CFTR correctors efficacy. Hepatology (Baltimore, Md.), 67(3), 972-988.

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