Dendritic cells (6–8 × 104) were seeded on fibronectin-coated Ibidi 8 well chambers and allowed to adhere for 1 hour at 37°C. Cells were rinsed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100, blocked with 0.2% normal donkey serum and stained overnight with KDEL, an ER marker (ADI-SPA-827-D; Enzo Life Sciences). The next day, cells were thoroughly washed, stained with an AlexaFluor488 (AF488) labelled donkey anti-mouse secondary antibody (A21202; Invitrogen) and Hoechst and mounted for microscopy. Confocal images were captured with a Zeiss LSM780 confocal microscope (Zeiss, Zaventem, Belgium). Images were taken by using a 63× Pln Apo/1.4 oil objective. Hoechst was excited with a Ti:Sa Mai Tai Laser at 790 nm and detected with a spectral bandwidth of 414–524 nm in front of the photomultiplier tube (PMT). AF488 was excited using the 488 line of a Multi Argon laser and detected with a spectral bandwidth of 495–579 nm in front of the PMT. The pinhole was set at 1 Airy Unit and 2x frame averaging was applied to reduce noise.
Adi spa 827 d
Manufactured by Enzo Life Sciences
The ADI-SPA-827-D is a laboratory equipment product manufactured by Enzo Life Sciences. It is designed for specific technical functions, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Additional information about the intended use or features of this product is not available.
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Lab products found in correlation
A21202, by Thermo Fisher Scientific (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
F7425, by Merck Group (1 mentions)
H 180, by Santa Cruz Biotechnology (1 mentions)
Sc 8035, by Santa Cruz Biotechnology (1 mentions)
H 160, by Santa Cruz Biotechnology (1 mentions)
A2228, by Merck Group (1 mentions)
Mms 101p, by Fortrea (1 mentions)
2 protocols using adi spa 827 d
1
Immunofluorescent Visualization of ER
2
Comprehensive Antibody Characterization Protocol
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We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R&D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) (Chen, 2006 (link)), rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).
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