MPs derived from bone marrow of WT or itgam−/− mice were seeded on 24-well plate coverslips. Adherent cells were infected with rBCG::PGL-I or rBCG::noPGL at MOI 5 for 30 min at 37°C. Cells were fixed in 4% PFA for 20 min. After saturation in D-PBS- BSA with 5% for 30 min, cells were labeled with anti-NFATc2 antibody (clone 25A10.D6.D2, Invitrogen) in PBS containing 0.1% Triton X-100, for 1 h at room temperature. After washings in PBS with 0.001% Triton X-100, cells were incubated with Alexa 633-conjugated goat anti-mouse IgG1 antibody (Invitrogen) for 1 h at room temperature. Slides were mounted with Fluoromount-G medium containing DAPI (Invitrogen). Images were captured with a confocal Leica TCS SP8 microscope. NFATc translocation was quantified by calculating the Manders coefficient using the JaCOP plugin (26 (link)) for Image J.
Fluoromount g medium
Manufactured by Thermo Fisher Scientific
Sourced in France
Fluoromount-G medium is a water-soluble, non-toxic mounting medium designed for use in fluorescence microscopy. It is a glycerol-based solution that helps preserve fluorescent signals in specimens.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (2 mentions)
Lab tek 2 cc2 8 well chamber slide system, by Thermo Fisher Scientific (2 mentions)
Tcs sp8 microscope, by Leica (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Anti panx1, by Merck Group (1 mentions)
Dmi 4000b confocal microscope, by Leica (1 mentions)
Ab130206, by Abcam (1 mentions)
Ab235178, by Abcam (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal scanning microscope, by Zeiss (1 mentions)
Cryostat, by Leica (1 mentions)
Triton x, by Merck Group (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Goat anti rabbit igg conjugated to alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab3080, by Merck Group (1 mentions)
Dii solution, by Thermo Fisher Scientific (1 mentions)
Isopentane, by Merck Group (1 mentions)
Zen 2010 b sp1, by Zeiss (1 mentions)
C11 bodipy581 591, by Merck Group (1 mentions)
12 protocols using fluoromount g medium
1
Quantifying NFAT Translocation in Mycobacteria-Infected Macrophages
2
Immunofluorescence Profiling of PANX1 in Neuroblastoma
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Neuroblastoma and peripheral nerve tissue array (MC602; US Biomax, Rockville, MD) was deparaffinized in xylene (2 washes, 5 minutes each) and rehydrated in graded alcohols (100% ethanol, 2 minutes; 95% ethanol, 2 minutes; 70% ethanol, 2 minutes; followed by two washes in deionized water, 5 minutes each). Antigen retrieval was performed using Tris-EDTA pH 9.0 at 95oC for 20 minutes. Following blocking in 2% bovine serum albumin (BSA) for one hour at room temperature, the tissue array was labeled with anti-PANX1 (Sigma-Aldrich, Cat#: HPA016930, 1:50) for one hour at 37 ºC and then washed 3 times in PBS, 10 minutes per wash at room temperature. After the washes, the tissue array was incubated with anti-rabbit secondary antibodies conjugated to Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, Cat#: A-11012, 1:200) for one hour at 37 ºC, followed by 3 washes with and a final wash in deionized water for 15 minutes. The background control was performed without incubating with anti-PANX1 antibodies. The slides were mounted using Fluoromount-G medium with DAPI (Invitrogen, Cat#: 00-4959-52) and visualized with the slide scanner Zeiss Axio Scan.Z1 (Toronto, ON, Canada). Quantification (mean fluorescence intensity) was done using ImageJ and averaged from three random field of the same area for each tumor core.
3
Immunofluorescence Staining Protocol
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Cells were grown on 12 mm round coverslips and fixed with 4% paraformaldehyde for 15 min, rinsed in PBS, incubated in NH4Cl 50 mM for 10 min, permeabilized with 0.5% Triton X100 for 15 min and blocked with 0.5% BSA/2% Normal Goat Serum for 10 min. Then, cells were incubated with primary antibodies at room temperature for 30 min. Slides were rinsed in PBS 0.5% BSA and incubated at room temperature in the dark for 30 min with secondary antibodies and rinsed in PBS 0.5% BSA. Finally, slides were washed in PBS, counterstained with Hoechst 33,342, and mounted in Fluoromount-G medium (Thermo Fisher Scientific). Images were digitally acquired with a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany).
4
Immunofluorescent Analysis of TGFBR1 and BMPR2
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After transfection or treatments, cells were seeded in a Nunc Lab-Tek II CC2 8-well Chamber Slide System at a density of 10,000/well and cultured for 2 days. Cells were fixed with 4% paraformaldehyde or with ice-cold methanol for 15 min. Samples were blocked in PBS/5% BSA/0.3% Triton X-100 for 1 h and incubated overnight at 4 °C with TGFBR1 (ab235178, 1/100, Abcam) or BMPR2 (ab130206, 1/100, Abcam). After washing in PBS, cells were incubated with Alexa Fluor™ 568 goat anti-mouse or anti-rabbit secondary antibodies (1/500, Life Technologies, Thermo Fisher Scientific). Slides were mounted using Fluoromount-G medium (#00-4958-02; Thermo Fischer Scientific). Images were acquired using a LEICA DMI 4000B confocal microscope (Leica Microsystems SA, Nanterre, France) with a 63× magnification oil-immersion objective. The intensity of fluorescence was measured using ImageJ software.
5
Immunofluorescence Staining of bEnd.3 Cells
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bEnd.3 cells were cultured in 8- or 16-well slide chambers (Tissue Tek). By the end of the experiment, cells were fixed in 4% freshly prepared ice-cold PFA for 10 min. Afterwards, they were washed 3 times with PBS and blocked in PBS containing 10% FCS for 30 min. The primary antibodies were applied for 1 h at RT in PBS containing 5% FCS and 0.1% Tween-20. After primary antibody incubation, cells were washed 3 times with PBS-Tween-20 (0.1%) and incubated with appropriate secondary antibodies diluted in PBS containing Hoechst (1:1000) for 1 h at RT. Finally, the cells were washed again three times with PBS and mounted using Fluoromount-G medium (Thermo Fischer Scientific).
6
Immunostaining and Imaging of Soleus Muscle
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Soleus muscles were isolated, pinned on a cork, embedded in OCT (VWR) and flash frozen in dry ice-cooled isopentane (Sigma). For immunofluorescence, 20 μm muscle sections were cut on a cryostat (Leica) and mounted on microscope slides. Slides were fixed for 20 min in 4% paraformaldehyde and washed in PBS before incubation with antibodies in 0.3% Triton-X (Sigma) in PBS plus 10% goat serum. Incubation with primary antibodies was performed overnight, whereas secondary antibody incubation was done over 2 h. Antibodies consisted of: rabbit anti-GFP IgG (Millipore AB3080, 1:1,000), mouse anti-dihydropyridine receptor α2-subunit (Sigma D218, 1:500) goat anti-rabbit IgG conjugated to Alexa Fluor 488 (Life Technologies A-11008, 1:500) and goat anti-mouse conjugated to Alexa Fluor 546 (Life Technologies A-21045, 1:500). Slides were mounted with fluoromount-G medium (Fischer Scientific, 0B100-01). For sarcolemma and T-tubule labelling, soleus muscles were isolated after trans-cardiac perfusion with 4% paraformaldehyde and post-fixed for 2 h at 4 °C, subsequently washed in PBS and incubated for 1 week in a 1 mg ml−1 DiI solution (Molecular Probes) in PBS at 4 °C (ref. 46 (link)). Stained muscles were washed with PBS, flash frozen and sectioned as described above. Images were acquired with a Zeiss LSM 710 confocal scanning microscope equipped with Zen 2009 software (Zeiss).
7
Lipid Peroxidation Imaging in Cells and Tissues
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We stained fixed cells with 2.5 µM C11‐BODIPY581/591 (SIGMA ALDRICH, D-3861) for 60 min. The ROS-dependent oxidation of the polyunsaturated butadienyl portion of this lipid probe results in a shift of the fluorescence emission peak from ~590 nm (reduced: red) to ~510 nm (oxidized: green)107 ,108 . Briefly, cells were treated with 0.2 µM FASNi or 0.2% DMSO, for 4 days. In rescue experiments, cells were co-treated with FASNi and one of the following: palmitate (100 µM), phosphatidylcholine (PC, 100 µM), or Ferrostatin-1 (Fer-1, 1 µM) for 4 days. 5 mM N-acyl-cysteine (NAC) was added for 60 min after 4 days of FASNi treatment. For snap-frozen samples, we used 10 µm-thick cryo-sections of A549 xenografts (5 mice/group) and lungs of CCSP-rtTA/Tet-op-Kras (3 mice/group). Samples were incubated with C11‐BODIPY581/591, washed with PBS, fixed with 10% formalin for 1 h, counterstained with DAPI and mounted using Fluoromount-G medium (Thermo Scientific). For cells, three images per slide were acquired using a Zeiss LSM 710 confocal microscope equipped with a Plan-Apo 63x/1.4 oil DIC M27 objective and ZEN 2010 B SP1 software (Zeiss). For tissues, three images per sample were acquired using a Lionheart FX (BioTek) and Gen5 software (v3.06). We quantified the images with ImageJ software (version 1.46; NIH, Bethesda, MD, USA).
8
EGFR Immunofluorescence Staining Protocol
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After treatment, cells were seeded in the Nunc Lab-Tek II CC2 8-well Chamber Slide System at a density of 2000 per well and cultured for 2 days. Cells were then fixed in ice-cold methanol for 10 min and washed in PBS. After a 60 min blocking step in PBS/3% BSA/0.3% Triton X-100, cells were incubated overnight at 4 °C with EGFR antibody (#4267; Cell Signaling Technology, Ozyme, Saint-Cyr-L’Ecole, France; dilution 1/50). After washing in PBS, cells were incubated with appropriate secondary antibodies (Life Technologies; dilution 1/500) and DAPI (#D9542; Sigma-Aldrich, St Quentin Fallavier Cedex, France; 1 µg/mL). After washing in PBS, the slides were mounted using Fluoromount-G medium (#00-4958-02; Thermo Fisher Scientific, Illkirch, France). Images were acquired using a LEICA TCS SPE II confocal microscope (Leica Microsystems SA, Nanterre Cedex, France), with a 60 × magnification oil-immersion objective, and analyzed with ImageJ software (https://imagej.nih.gov , access on 3 May 2021).
9
Cytokine Quantification and NF-κB Activation
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Quantification of cytokines present in cell culture supernatants was performed by ELISA using the DuoSet ELISA kit (R&D Systems) for TNF-α, IL-β and IL-6, according to the manufacturer’s instructions.
Analysis of NF-ΚB activation was performed at 6 hr post-infection. Cells were fixed with 4% paraformaldehyde at RT for 15 min, incubated for 5 min with 0.5% saponin (Sigma-Aldrich) in PBS and then incubated for 30 min in 1% BSA (Sigma-Aldrich) and 0.1% saponin (Sigma-Aldrich) in PBS, to permeabilize the cells and to block nonspecific binding. Cells were incubated with anti-NF-κB antibody (Thermo Fisher) overnight at 4°C. Cells were washed and incubated with Alexa Fluor 555 secondary antibody (Thermo Fisher) for 2 hr. Nuclei were stained with DAPI (1 μg/ml) for 10 min. After labeling, coverslips were set in Fluoromount G medium (Thermo Fisher). Fluorescence was analyzed using a Leica TCS SP8 Confocal System and the NF-κB translocation analysis was performed using Icy software.
Analysis of NF-ΚB activation was performed at 6 hr post-infection. Cells were fixed with 4% paraformaldehyde at RT for 15 min, incubated for 5 min with 0.5% saponin (Sigma-Aldrich) in PBS and then incubated for 30 min in 1% BSA (Sigma-Aldrich) and 0.1% saponin (Sigma-Aldrich) in PBS, to permeabilize the cells and to block nonspecific binding. Cells were incubated with anti-NF-κB antibody (Thermo Fisher) overnight at 4°C. Cells were washed and incubated with Alexa Fluor 555 secondary antibody (Thermo Fisher) for 2 hr. Nuclei were stained with DAPI (1 μg/ml) for 10 min. After labeling, coverslips were set in Fluoromount G medium (Thermo Fisher). Fluorescence was analyzed using a Leica TCS SP8 Confocal System and the NF-κB translocation analysis was performed using Icy software.
10
Sperm Storage in Uterovaginal Junction
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Hens were slaughtered 24 h after the second insemination, and reproductive tracts were isolated (Supplementary Data 1 A). The villi (n = 5 per animal) containing SST distributed in the uterovaginal junction (UVJ ) (Supplementary Data 1 B and 1 C) were randomly dissected from the underlying mucosa. Pieces of villi were fixed in a 4% paraformaldehyde solution at 37°C for 30 min and then mounted on microscope slides using Fluoromount-G medium (ThermoFisher). Images were acquired using a microscope slide scanner (Axio Scan.Z1, Zeiss) with a 20 × objective lens. Fluorescence imaging was performed using the X-Cite Illumination System. The emission Band Pass filters used were EM BP 445/50 (DAPI) for Hoechst-33342 labeled sperm and EM BP 690/50 (Alexa Fluor 633) for SST autofluorescence. The presence of sperm-filled SST and sperm-empty SST in each UVJ villus was identified manually using QuPath image analysis software and the percentage of SST filled with stained sperm was determined.
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