The MCF-7/WT and MCF-7/DOX-1 cells were fixed onto glass coverslips by 4% paraformaldehyde (P6148, Merck, Germany) for 10 min, and subsequently stained with either monoclonal antibody anti-CD24 conjugated to Milli-Mark™ fluorescein isothiocyanate (FCMAB188F, anti-CD24–FITC; clone SN3, 1:15 (v/v) dilution, EMD Millipore Corporation, Burlington, MA, USA) or monoclonal antibody anti-human CD44 conjugated to R-phycoerythrin (CBL154P, anti-CD44–R-PE; clone F10-44-2, dilution 1:15, EMD Millipore Corporation, Burlington, MA, USA) at room temperature (RT) for 1 h. After washing with PBS (524650, Merck, Germany) containing 0.05% Tween-20 (P9416, Merck, Germany), the cell nuclei were stained with Hoechst 34580 (H21486, 2 µg/mL, Thermo Fisher Scientific, USA) for 10 min. The cells were visualized using a confocal microscope Zeiss LSM 5 DUO (Zeiss, Germany) at λexc = 488 nm and λem = 515–550 nm for anti-CD24-FITC, at λexc = 560nm and λem > 573 nm for anti-CD44-R-PE, and at λexc = 405 nm and λem = 420–480 nm for Hoechst 34580. The photomicrographs were scanned using a 63× objective.
Hoechst 34580
Manufactured by Zeiss
Sourced in Germany
Hoechst 34580 is a cell-permeable, fluorescent dye that binds to DNA. It is commonly used in laboratory applications to stain and visualize cellular nuclei.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using hoechst 34580
1
Immunofluorescence Analysis of CD24 and CD44
2
Immunofluorescent Detection of Extracellular Matrix Components
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The samples were fixed with frozen 70% methanol, washed with PBS and incubated in 3% FBS in PBS/0.1% Triton for 30 min at RT. Primary monoclonal antibody against type I collagen (dilution 1:20, M-38c, DSHB, USA) or osteocalcin (dilution 1:20, osteocalcin 1-49 [human] IgG, T4743, Peninsula Laboratories, USA) was then added overnight at 2-8°C. After three washes with PBS/0.05%, and once in PBS, the samples were incubated with secondary antibody Alexa Fluor 488-conjugated antimouse antibody (dilution 1:300, 45 min, RT, A10667, Life Technologies) or Alexa Fluor 633-conjugated anti-rabbit antibody (dilution 1:300, Life Technologies), cell nuclei were stained with propidium iodide (5 μg/ml in PBS, 5 min, RT, Sigma-Aldrich) or using Hoechst 34580 (30 min at RT, dilution 1:5000, H21486, Life Technologies). The cells were visualized using a confocal microscope ZEISS LSM 5 DUO at λexc = 488 nm and λem = 505-550 nm for Alexa Fluor 488, and λex = 560 nm, λem >575 nm for propidium iodide), λexc = 405 nm and λexc = 420-650 nm for Hoechst 34580, λexc = 633 nm and λem >650 nm for Alexa Fluor 633. At least eight photomicrographs per group were analyzed for the intensity measurement from three samples and two negative controls per group.
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