For the detection of NUDT4 and HDAC4, transfected PANC-1 and BxPC-3 cells were incubated in the coverslips of six-well plates at 37 °C overnight. The cells were washed with PBS twice, fixed with 4% formaldehyde and then permeabilized with 0.5% Triton (9036–19-5, Sigma-Aldrich). The treated cells were incubated with primary antibody overnight at 4 °C and then with secondary antibody. Sample mounting and nuclear staining were performed using ProLong™ Gold Antifade Mountant with DNA Stain DAPI (P36935, ThermoFisher). The samples stained only with secondary antibody were set as negative control. IF images were obtained using a fluorescence microscope (DMi8, Leica, Germany). Fluorescence intensity analysis was the same with FISH assay. The primary and secondary antibodies are listed in Supplementary Table 2 .
Prolong gold antifade mountant with dna stain dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong™ Gold Antifade Mountant with DNA Stain DAPI is a ready-to-use mounting medium for fluorescence microscopy. It contains the DNA-binding dye DAPI, which fluoresces when bound to DNA, allowing for the visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Fluorescein isothiocyanate conjugated anti mouse secondary antibody, by Merck Group (1 mentions)
Confocal microscope, by Leica (1 mentions)
Anti cd8, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by R&D Systems (1 mentions)
Anti granzyme b, by Thermo Fisher Scientific (1 mentions)
Opal reagents, by Akoya Biosciences (1 mentions)
Alexa fluor 647 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Alexa fluor plus 555 conjugated donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde solution, by Santa Cruz Biotechnology (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Sucrose, by Merck Group (1 mentions)
4 protocols using prolong gold antifade mountant with dna stain dapi
1
Detecting NUDT4 and HDAC4 in PANC-1 and BxPC-3 cells
2
Nrf2 Immunofluorescence in HaCaT Cells
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HaCaT cells (3 × 105 cells/mL/well) were seeded into an eight-well chamber containing DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) P/S and incubated at 37 °C for 16 h. After treatment with CSK or H2O2, the cells were fixed using a 4% formaldehyde solution and permeabilized using 0.2% TritonX-100 (Sigma, St. Louis, MO, USA). Subsequently, the cells were blocked with 3% bovine serum albumin and then incubated with an anti-Nrf2 antibody (1:50 in 3% bovine serum albumin) for 2 h at room temperature. Then, fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Sigma, St. Louis, MO, USA) was added, and the cells were incubated for 2 h at room temperature. The cells were mounted onto the cover glass, using ProLong Gold Antifade Mountant with DNA Stain DAPI (Thermo Fisher, Waltham, MA, USA), and observed and photographed using a confocal microscope (Leica, Wetzlar, Germany). The exposure time was consistent across samples in all experiments.
3
Multi-Epitope Immunofluorescence Profiling
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Immunofluorescence staining utilized tyramide signal amplification staining performed using OPAL Reagents (Akoya Biosciences, Inc, Marlborough, MA). Briefly, tumor tissues were formalin fixed and embedded in paraffin blocks and cut into 10 μm sections at the Cedars-Sinai Biobank and Research Pathology Core. Tissue sections were deparaffinized and rehydrated, then antigen retrieval was performed in Tris-EDTA or citrate buffer. Primary antibodies anti-CD8 (ebioscience, 4SM15), anti-CD4 (R&D, GK1.5), anti-CD90 (Sino Biological, Cat #50461-T44), anti-CD73 (Sino Biological, Cat # 50231-T56), anti-GranzymeB (ebioscience, 16G6), anti-CD90.1 (BioLegend, OX-7), anti-CD45.1 (Invitrogen, A20) diluted to 1:200 were incubated overnight at 4°C. Secondary antibodies conjugated to HRP-polymers (Abcam, Cambridge, UK) were incubated for 15 min and then washed before slides were incubated with OPAL fluorophores (OPAL 520, OPAL, 650, OPAL 570) diluted 1:200 for 10 min at RT. Slides were washed in PBST before performing subsequent rounds of antigen retrieval and staining. Tissues were mounted with ProLong Gold Antifade Mountant with DNA Stain DAPI (Invitrogen). Images were acquired on ECHO Revolution upright microscope equipped with sCMOS Mono camera.
4
Immunofluorescent Characterization of Teratomas
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Teratomas were harvested and fixed in 4% paraformaldehyde solution (Santa Cruz Biotechnology, Inc.) for 24hr at room temperature then washed with PBS 3 times and stored in PBS at 4°C until ready for use. Samples were cryoprotected with sucrose (Sigma-Aldrich), cryopreserved in Tissue-Tek O.C.T. compound (Sakura), and sectioned at 5µm with a Leica CM3050 S cryostat. Sections were incubated with PBS containing 0.1% triton X-100 (Sigma-Aldrich) and 5% donkey serum (Jackson ImmunoResearch). Sections were incubated overnight with unconjugated goat anti-mouse/rat CD47 N-terminal IgV-like extracellular domain antibody (R&D Systems), biotin-conjugated anti-mouse CD55 REAfinity antibody (clone REA300, Miltenyi Biotec), unconjugated rabbit anti-mouse CD59a antibody (clone 108, SinoBiological), and AF594-conjugated anti-human mitochondria antibody (clone 113-1, Novus Biologicals) followed by a 1hr incubation with Alexa Fluor 647-conjugated AffiniPure donkey anti-goat IgG antibody (Jackson ImmunoResearch), Alexa Fluor 647-conjugated streptavidin (Jackson ImmunoResearch), or Alexa Fluor Plus 555-conjugated donkey anti-rabbit IgG antibody (Invitrogen). Slides were mounted with ProLong Gold Antifade Mountant with DNA stain DAPI (Invitrogen) and imaged on a Zeiss LSM 800 confocal laser scanning microscope. The resulting images were processed and analyzed with Fiji (ImageJ).
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