Animals exposed to red NPs were euthanized by tricaine overdose, fixed in 4% PFA for 24 h at 4°C, then immersed in 30% sucrose for several days, embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek USA), flash frozen in isopentane, and sectioned using a CM3050 S cryostat (Leica). DCs were stained using 1:50 FITC conjugate peanut agglutinin (FITC-PNA) (US Biological). Macrophage, neutrophil, and IgZ+ were stained using 1:250 rabbit anti-Mpeg1, 1:50 rabbit anti-Mpx, and 1:500 rabbit anti-IgZ-IN2 antibodies (AnaSpec), respectively, and 1:250 cross-adsorbed goat anti-rabbit secondary antibody (Thermo Fisher), which were either conjugated to DyLight 488 (for IgZ staining) or to DyLight 633 (for Mpx and Mpeg1 stainings). For double staining, cryosections were saturated with 5% BSA between FITC-PNA and antibody labeling. No cross staining was observed for PNA/IgZ labeling, while a weak PNA signal could be detected in a few Mpeg1+ macrophages. Mpx+ neutrophils consistently displayed moderate PNA signal, but distinct from DCs, which displayed high granular intracytoplasmic PNA staining and were Mpx−. Cryosections were co-stained with DyLight 488-Phalloidin (Thermo Fisher) and DAPI (Euromedex) and analyzed using a SP5 upward confocal microscope (Leica) with 63×/1.4NA objective and ImageJ.
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Euromedex
Sourced in France
DAPI is a fluorescent stain that binds to DNA. It can be used to visualize nuclei in cells.
Automatically generated - may contain errors
Lab products found in correlation
Dm4000b, by Leica (2 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (2 mentions)
Oil red o, by Merck Group (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Dutscher (2 mentions)
Goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dylight 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Bodipy fl c12, by Thermo Fisher Scientific (1 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti β actin polyclonal antibody, by ABclonal (1 mentions)
Goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Cm 3000, by Leica (1 mentions)
8 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Multicolor Immune Cell Labeling
2
Immunofluorescence Analysis of TLR Signaling Pathway
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After infection, cells were fixed by immersion in 4% paraformaldehyde in PBS. Sections were incubated with primary Ab against TLR2 (1:250), TLR4 (1:250), MyD88 (1:500), MAL (1:500), TRIF (1:500), TRAM (1:500), SARM (1:600), and SOCS-1 (1:600), respectively, at 4°C for 24 h. After incubation, sections were incubated with the secondary Ab for 1 h at room temperature (1:250 to 1:500 of Alexa Fluor 594 or Alexa Fluor 488 (Invitrogen, Thermo Fisher, Illkirch, France)). Nuclei were stained with DAPI 200 nM (Euromedex, Souffelweyersheim, France) and β-actin was labeled with phalloïdin Alexa Fluor 546 (Thermo Fisher) in GEC. Finally, slides were mounted (Dako, Trappes, France) and observed with a fluorescence microscope (Leica DM4000B).
3
Cell Culture and Lipid Staining
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The human breast cancer cell, MCF-7, obtained from American Type Culture Collection (ATCC, LGC Standards, Molsheim, France), was maintained in DMEM medium (Dulbecco modified Eagle’s medium) supplemented with 10% (v/v) FBS (fetal bovine serum), while the SUM159PT cell line (provided by Philippe Benaroch, Cellular transport and immunity team, Curie Institute, Paris, France), was maintained in Ham’s F12 medium supplemented with 5% heat inactivated FBS, 10 mM HEPES, 1 µg/mL hydrocortisone and 5 µg/mL of human insulin. The hTert-immortalized HMEC cell line (provided by Anne-Pierre Morel, EMT and Cancer Cell Plasticity team, Léon Bérard Center, Lyon, France) was maintained in DMEM/F12 medium supplemented with 10% FBS, 10 ng/mL hEGF (human epidermal growth factor), 0.5 µg/mL hydrocortisone, 10 µg/mL insulin, 0.5 µg/mL puromycin. All cell lines were maintained at 37 °C in a humidified atmosphere with 5% CO2.
All reagents were purchased from the indicated manufacturers and prepared according to manufacturer’s instructions and used at the following concentrations: Oil Red O: 0.6% (Sigma-Aldrich, Saint-Louis, MO, USA), SSO: 150µM (AbCam, Cambridge, UK), BODIPY 493/503 (Thermofischer, Waltham, MA, USA), 1 µM for microscopy imaging and 10 nM for flow cytometry, BODIPY-FL C12 (Thermofischer), 20 µM, DAPI (Euromedex, Souffelweyersheim, France), 1 µM.
All reagents were purchased from the indicated manufacturers and prepared according to manufacturer’s instructions and used at the following concentrations: Oil Red O: 0.6% (Sigma-Aldrich, Saint-Louis, MO, USA), SSO: 150µM (AbCam, Cambridge, UK), BODIPY 493/503 (Thermofischer, Waltham, MA, USA), 1 µM for microscopy imaging and 10 nM for flow cytometry, BODIPY-FL C12 (Thermofischer), 20 µM, DAPI (Euromedex, Souffelweyersheim, France), 1 µM.
4
Cell Culture and Antibody Staining Protocol
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Vero (clone E6) and HEK-293T cells were cultured at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) growth medium supplemented with 5 to 10% of heat-inactivated fetal bovine serum (Dutscher, Brumath, France) and antibiotics. Mouse anti-flavivirus E mAb 4G2 was purchased from RD-Biotech (Besançon, Fance). Mouse anti-DDDDK tag (binds to FLAG tag sequence) and mouse anti-6 × (His) mAbs were purchased from Abcam (Birmingham, UK). Donkey anti-mouse IgG, HRP conjugate, horseradish peroxidase (HRP) was purchased from ImmunoReagents (Raleigh, USA). Donkey anti-mouse IgG, Alexa Fluor 488 conjugate, was purchased from Thermo Fisher Scientific (Les Ulis, France). DAPI was purchased from Euromedex (Souffelweyersheim, France).
5
Antibody Characterization for Viperin
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Mouse anti-viperin antibody clone MaP.VIP was purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). The mouse anti-DDDDK tag mAb (anti-FLAGtag antibody), goat anti-mouse and goat anti-rabbit immunoglobulin-horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Abcam (Cambridge, UK). DAPI was purchased from Euromedex (Souffelweyersheim, France). The rabbit anti-α tubulin polyclonal Ab was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).
6
Culturing HEK-293T and Huh7 cells
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Human embryonic kidney HEK-293T cells (ATCC, CRL-1573) were cultured in minimum essential media (MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS) and human hepatoma Huh7 cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Dutscher, Brumath, France). Both cell lines were cultured with antibiotics (PAN Biotech Dutscher, Brumath, France) at 37 °C under a 5% CO2 atmosphere. Mouse anti-DDDDK tag monoclonal antibody (FLAG Ab) and mouse anti-6x(His) monoclonal antibody was purchased from Abcam (Cambridge, UK). The mouse anti-flavivirus NS1 antibody [D/2/D6/B7] (NS1 mAbD/2/D6/B7), which reacts with a conformational epitope present on DENV-2, was purchased from Abcam (Cambridge, UK). The rabbit dengue virus type 2 NS1 polyclonal antibody PA5-33207 (NS1 AbPA5-32207) raised against the residues NS1-117/301 was purchased from (Thermo Fisher Scientific, Les Ulis, France). The rabbit anti-β actin polyclonal antibody was purchased from ABclonal (Massachusetts, USA). Goat anti-mouse IgG secondary antibody and Alexa Fluor Plus 488, was purchased from Invitrogen (Thermo Fisher, Les Ulis, France). Anti-mouse and anti-rabbit IgG HRP-conjugated secondary antibodies were purchased from Abcam (Cambridge, UK). DAPI was purchased from Euromedex (Souffelweyersheim, France).
7
Culturing Breast Cancer Cell Lines
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The human breast cancer cell lines MCF-7 obtained from American Type Culture Collection (ATCC, LGC Standards, France) was maintained in DMEM medium supplemented with 10% (v/v) FBS (fetal bovine serum) while the SUM159PT cell line was maintained in Ham's F12 medium supplemented with 5% heat inactivated FBS, 10 mM HEPES, 1 µg/ml hydrocortisone and 5 µg/ml of human insulin. The hTert-immortalized HMEC cell line was maintained in DMEM/F12 medium supplemented with 10% FBS, 10 ng/ml hEGF ( HER2 overexpression -0 growth factor), 0,5 µg/ml hydrocortisone, 10 µg/ml insulin, 0,5 µg/ml puromycin. All cell lines were maintained at 37 °C in a humidified atmosphere with 5% CO2. All reagents were used at the following concentrations: Oil Red O: 0.6% (Sigma-Aldrich), SSO: 150µM (AbCam), BODIPY 493/503 (Thermofischer): 1 µM for microscopy imaging, DAPI (Euromedex): 1 µM.
8
Histological Characterization of Implants
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For histology, after 2 weeks implantation, some implants were fixed for 24 h in Bouin-Hollande and embedded in paraffin. Serial sections (7 µm) were stained with Mallory's stain. Sections were observed in a Leica DM4000B microscope. Other implants were embedded in Tissue-Tek, frozen at -20°C and sectioned (10µm) using a cryostat (Leica, CM3000). Serial sections were rinsed with PBS and fixed for 10 min with 4% paraformaldehyde at 4°C. After washing three times for 5 min in PBS at room temperature, sections were incubated for 30 min at room temperature in a blocking solution of 1% bovine serum albumin (BSA) and 0.1% Triton X100 and then incubated for one night at 4°C with the primary antibodies (Table 1). After washing with PBS, sections were incubated with secondary antibodies (Molecular Probes, Invitrogen) (Table 1). Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI, Euromedex, Souffelweyersheim, France). Negative controls were performed either omitting the primary antibody or using normal goat serum (NGS). After 3 additional washes in PBS, slides were mounted in fluorescence mounting medium (Dako, Trappes, France) and observed with a fluorescence microscope (Leica DM4000B).
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