Cells seeded on coverslips were treated as indicated and fixed in ~4% formaldehyde (Sigma). Next, the cells were permeabilized in 0.1% Triton X-100 and stained with indicated antibodies and Hoechst 33342 (Thermo Fisher Scientific). The coverslips were mounted in ProLong Diamond Antifade Mounting (Thermo Fisher Scientific) reagent and examined with a ×63 objective on a Zeiss confocal Laser Scanning Microscope (LSM) 880 (Jena, Germany). Images were prepared with Fiji Image J software and Adobe Illustrator CS4 14.0.0.
Prolong diamond antifade mounting
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
ProLong Diamond Antifade Mounting is a mounting medium designed for the preservation and protection of fluorescently labeled samples. It is intended to be used with fluorescent microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Illustrator cs4 14, by Adobe (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Opal 7 color automation ihc kit, by Akoya Biosciences (1 mentions)
Superfrost plus, by Avantor (1 mentions)
Anti ki 67 clone 30 9, by Roche (1 mentions)
Anti cd20 clone l26, by Roche (1 mentions)
Anti cd3 clone 2gv6, by Roche (1 mentions)
Quant it picogreen dsdna reagent, by Thermo Fisher Scientific (1 mentions)
6 well culture plate, by Corning (1 mentions)
Lsm 710 duo, by Zeiss (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti tcf4, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
4 protocols using prolong diamond antifade mounting
1
Immunofluorescence Staining of Cells
2
Multiplex Immunohistochemistry for Tissue Analysis
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Tissue sections were prepared from formalin-fixed paraffin embedded tissue blocks and cut to 4 μm serial sections and mounted on Superfrost Plus (VWR). The procedure for multiplex immunohistochemistry (mIHC) was followed by a manufacturer’s protocol for Opal7-color automation IHC kit (Akoya Bioscience), and the staining was performed with Autostainer DISCOVERY ULTRA (Ventana). Antibodies used in mIHC are anti-CD3 (clone 2GV6, Ventana), anti-CD20 (clone L26, Ventana), anti-Ki67 (clone 30-9, Ventana), anti-FOXP3 (clone SP97, Spring), anti-pan cytokeratin (CK; clone AE1/AE3, DAKO), anti-CD117 (clone c-kit, DAKO). The molecular markers of immune panel (CD3, CD20, Ki67, CKs, FOXP3, and CD117) were visualized with Opal520, Opal540, Opal570, Opal620, Opal650, and Opal690, respectively. DAPI counterstaining was performed with Discovery QD DAPI (Roche). ProLong Diamond Antifade Mounting (ThermoScientific) was used for mounting the coverslip. Detailed staining conditions and autostainer’s protocols are reported in our recent report86 (link).
3
DNA Tagging and Cell Imaging Protocol
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For DNA tagging, 500 µl SF was stained with the Quant-iT PicoGreen dye at a ratio of 1:200 using Quant-iT stock (cat. no. P7581; Quant-iT™ PicoGreen™ dsDNA Reagent; Invitrogen; Thermo Fisher Scientific, Inc.), and incubated 30 min at 37 °C. Subsequently, the stained SF was added and incubated at different times (≤ 80 min) to NHI3T3 cells cultured for 24 h over slides in 6-well culture plates (Corning, Inc.). Probes with PBS were used as controls. After exposure, cells were washed three times with 500 µl PBS, mounted with Prolong Diamond Antifade Mounting (Molecular Probes; Thermo Fisher Scientific, Inc.), and analyzed with a confocal microscope (LSM 710 DUO; Zeiss GmbH) with lasers exhibiting excitation wavelengths at 488 and 594 nm. In total, 20 fields were observed for each treatment, and representative images were acquired. The data from three independent experiments were collected with a 63X objective oil immersion lens.
4
Immunofluorescence Analysis of Protein Localization
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C33A and HaCaT cells were seeded over slides in 6 well plates and transfected with the indicated plasmids. After 48 h post-transfection cells were fixed with 3.7% paraformaldehyde in PBS for 10 min and permeabilized with PBS-0.1% Triton X-100. Then, cells were incubated with anti-FLAG M2 (Sigma Aldrich, Sant Louis, MO, USA) and anti-β-catenin (Cell Signaling) or anti-TCF-4 (Santa Cruz Biotechnologies, Dallas, TX, USA) antibodies overnight at 4 °C, after blocking with a 0.3% BSA solution. Cells were washed extensively with PBS and later incubated with anti-rabbit or anti-mouse antibodies conjugated to Rhodamine or Alexa-488 (Invitrogen, Carlsbad, CA, USA), respectively. Slides were washed and mounted with Prolong Diamond Antifade Mounting (Molecular Probes, Eugene, OR, USA) and then analyzed with a confocal microscope (Zeiss LSM 710 DUO, Oberkochen, Germany), with lasers giving excitation lines at 488 and 594 nm. Around twenty fields were observed for each treatment and representative images were acquired. The data of three independent experiments were collected with a 63× objective oil immersion lens.
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