Cells were grown on glass coverslips overnight and were either treated with DMSO or MG132 for 4 hours. The samples were fixed with 2% paraformaldehyde (in PBS, pH 7.2) for 20 minutes. Samples were then washed 3x with PBS and blocked with 2% BSA with 0.1% Triton X-100 in PBS for 1 hour. Anti-c-MYC primary antibody (1∶200, Millipore #06-340) was incubated with 1% BSA and 0.05% Triton X-100 in PBS overnight in a humidified chamber. The samples were washed 3x with PBS. Alexa Fluor 568 donkey anti-rabbit (1∶400, Life Technologies) was diluted in 1% donkey serum with 0.05% triton X-100 in PBS and incubated with the samples for 1 hour. Samples were washed 3x with PBS and mounted on glass slides with Duolink In Situ mounting medium with DAPI (Sigma). Images were acquired on a Zeiss LSM700 Confocal Microscope.
Duolink in situ mounting medium with dapi
Manufactured by Merck Group
Sourced in United States
Duolink In Situ Mounting Medium with DAPI is a laboratory product used for mounting and preserving fluorescently labeled samples. It contains the nuclear stain DAPI, which enables the visualization of cell nuclei.
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Lab products found in correlation
Duolink in situ pla probe anti mouse minus, by Merck Group (4 mentions)
Duolink in situ pla probe anti rabbit plus, by Merck Group (4 mentions)
Duolink in situ detection reagents red, by Merck Group (3 mentions)
Duolink blocking solution, by Merck Group (3 mentions)
Lsm 780, by Zeiss (3 mentions)
Axiovert fluorescent microscope, by Zeiss (3 mentions)
Nunc lab tek glass chamber slide system, by Thermo Fisher Scientific (2 mentions)
Motorized inverted research microscope model ix81, by Olympus (2 mentions)
Duolink in situ pla probe, by Merck Group (2 mentions)
Duolink in situ detection reagents red kit, by Merck Group (2 mentions)
Duolink in situ pla technology, by Merck Group (2 mentions)
Duolink in situ detection reagents orange, by Merck Group (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Duolink in situ proximity ligation assay, by Merck Group (1 mentions)
Lab tek, by Thermo Fisher Scientific (1 mentions)
Vegf165, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Matrigel membrane matrix growth factor reduced, by Corning (1 mentions)
38 protocols using duolink in situ mounting medium with dapi
1
Immunofluorescence analysis of c-MYC
2
Proximity Ligation Assay for Protein Interactions
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Proximity ligation assay (PLA) by indirect detection was done using Duolink reagents and instructions (Sigma-Aldrich) on rat brain cryosections (16 μm) (see ‘Tissue preparation’ section). After incubation in Duolink blocking solution, the tissue sections were incubated with anti-TRPV4 (LS-Bio #LS-C94498, 1:400) and anti-NKCC1 (Dundee University #S022D, 2 µg/ml) overnight at 4 °C. The secondary antibodies were conjugated to a MINUS and a PLUS PLA probe, and were detectable as fluorescent speckles only upon close contact [47 (link)]. A technical control omitting both of the primary antibodies was included. Ligation and amplification of the samples were done according to the Duolink protocol, the sections were mounted with coverslips using the Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich). Micrographs were acquired with a Zeiss LSM700 point laser (Argon Lasos RMC781272) scanning confocal microscope with a Zeiss Plan-Apochromat 20×/numerical aperture (NA) 1.6 oil objective (Carl Zeiss, Oberkochen). All micrographs were sampled in a frame scan mode.
3
ErbB2-Nucleolin Interaction Visualization
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For PLA cells were plated in 16-well Nunc Lab-Tek glass Chamber Slide System (178599; Thermo Scientific) and treated as indicated for 2 days. Following fixation, cells were incubated with rabbit anti-ErbB2 and mouse anti-nucleolin antibodies. PLA was performed using the Duolink In-Situ PLA probes: anti-rabbit MINUS and anti-mouse PLUS, and the Duolink In-Situ Detection Reagents Red kit (DUO92005; DUO92001; DUO92008, respectively; Sigma-Aldrich), according to the manufacturer’s instructions. Nuclei were stained using the Duolink In-Situ Mounting Medium with DAPI (DUO82040; Sigma-Aldrich). Slides were visualized 24 h post staining and images were obtained using an Olympus motorized inverted research microscope Model IX81 (×60 magnification). Signal intensity was determined using ImageJ software.
4
Proximity Ligation Assay for VDAC1-IP3R3
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PLA was performed using Duolink In Situ Red Started Kit Mouse/Rabbit (Duolink In Situ Detection Reagents Red, Sigma-Aldrich, DUO92008; Duolink In Situ PLA Probe Anti-Mouse MINUS, Sigma-Aldrich, DUO92004; Duolink In Situ PLA Probe Anti-Rabbit PLUS, Sigma-Aldrich, DUO92004; Duolink In Situ Mounting Medium with DAPI, Sigma-Aldrich, DUO82040) accordingly to the manufacturer’s instructions. Briefly, after fixation with 4% PFA and permeabilization with 0.1% Triton, HeLa cells were incubated with primary antibody, VDAC1 (rabbit; ab15895) and IP3R3 (mouse; BD Bioscience 610312), in blocking solution (PBS + 0.1% Triton X-100 + 4% BSA) ON. Secondary antibodies PLUS and MINUS (anti-rabbit and anti-mouse IgG antibodies conjugated with oligonucleotides) were incubated 1:5 in blocking solution for 1 h at 37°C. The ligation solution (ligation buffer 1:5, ligase 1:40 in MQ) was then incubated for 30 min at 37°C. The amplification solution (amplification buffer 1:5, polymerase 1:40 in MQ) was incubated for 1 h 40 min at 37°C. After incubation, slides were mounted with Duolink In Situ Mounting Medium with DAPI. As a negative control, we incubated VDAC1 alone and with both PLUS and MINUS secondary antibodies.
5
Visualizing VDAC1-IP3R Interactions Using Duolink PLA
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Duolink in situ Proximity Ligation Assay (Sigma Aldrich, USA) permits the detection of protein–protein interactions. JEG3 cells treated with and without CER 16:0 (20 μM) and 2-OE (25 μM) for 6 h, were cultured on eight-well chamber slides (LabTek, ThermoFisher, CA). Cells were then washed with PBS, fixed with cold 1:1 methanol and acetone for 3 min and permeabilized with 0.2% Triton X-100 for 5 min. Following a blocking step with Duolink blocking solution for 30 min at 37 °C, cells were incubated with VDAC1 and IP3R antibodies overnight at 4 °C. Hybridization of antibodies using plus and minus PLA probes raised against species of respective primary antibodies, ligation, and amplification reactions were performed according to the manufacturer's protocol. Slides were mounted with Duolink in situ mounting medium with DAPI (Sigma Aldrich, USA), and pictures were obtained using spinning disc confocal microscope with Volocity Imaging system.
6
Angiogenesis Evaluation Protocol
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PMX disodium hemipentahydrate was purchased from Shipla Medicare (Karnataka, India). Caprylocaproyl macrogol-8-glycerides (Labrasol) were provided by Gattefossé (Saint Priest, France). Deoxycholic acid (DOCA), Drabkin’s reagent, and Duolink in situ mounting medium with DAPI were obtained from Sigma-Aldrich (St. Louis, MO, USA). Polyoxyethylene (160) polyoxypropylene (30) glycol (poloxamer 188; P188) was obtained from BASF (Ludwigshafen, Germany). Matrigel membrane matrix (growth factor reduced) was purchased from Corning (Manassas, VA, USA). VEGF165 and basic fibroblast growth factor (bFGF) were purchased from Pepro Tech (Rocky Hill, NJ, USA). Brij-35 solution was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The solvents used in high-performance liquid chromatography (HPLC) were obtained from Merck and Thermo Fisher Scientific (Waltham, MA, USA). Polyethylene glycol-conjugated rat anti-mouse CD-31 antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-thrombospondin-1 antibody and Goat anti-rabbit Alexa Fluor 488 were purchased from Abcam (Cambridge, UK) and Invitrogen (Carlsbad, CA, USA), respectively.
7
Proximity Ligation Assay for ErbB2-Nucleolin
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For proximity ligation assay (PLA) cells were plated in 16-well Nunc Lab-Tek glass Chamber Slide System (178599; Thermo Scientific) and grown for 48-72h with or without siRNA treatment, as indicated. Following fixation, cells were incubated with rabbit anti-ErbB2 and mouse anti-nucleolin antibodies. PLA was performed using the Duolink In-Situ PLA probes: anti-rabbit MINUS and anti-mouse PLUS and the Duolink In-Situ Detection Reagents Red kit (DUO92005; DUO92001; DUO92008, respectively; Sigma-Aldrich), according to the manufacturer's instructions. Nuclei were stained using the Duolink In-Situ Mounting Medium with DAPI (DUO82040; Sigma-Aldrich). Slides were visualized 24h post-staining and images were obtained using an Olympus motorized inverted research microscope Model IX81 (60× magnification). Signal intensity was determined using ImageJ software.
8
Proximity Ligation Assay for Protein Interactions
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For visualization of protein–protein interaction in situ, Proximity Ligation Assay (PLA) was performed according to manufacturer's instructions (Duolink using PLA Technology, Sigma). Briefly, 48 h after transient HA-TET1 and/or myc-GADD45a and myc-NFYC (control protein) transfection, HEK293T cells were fixed in 100% MeOH (Sigma) for 10 min at −20 °C, permeabilized with 0.5% TritonX100 (Sigma) for 10 min, blocked and incubated with primary antibodies anti-HA (Abcam) and anti-myc (Sigma) (both diluted 1:1000) overnight at 4 °C. Then, cells were incubated with respective secondary antibodies (Duolink In Situ PLA Probes Anti-Mouse and Anti-Rabbit both PLUS and MINUS, SIGMA) followed by a ligation and subsequent amplification step to visualize protein interaction by red fluorescence signals (Duolink In Situ Detection Reagents Red, Sigma). To visualize individual proteins by Self-PLA, the respective primary antibody was combined with two secondary antibodies (PLA probes both PLUS and MINUS). Nuclei were counterstained by DAPI-containing mounting medium (Duolink In Situ Mounting Medium with DAPI, Sigma) and analysis was performed using a TCS SP5 confocal microscope (Leica) and 63× oil immersion objective lens. The speckles are likely limited to a fraction of cells due to incomplete transfection efficiency.
9
Visualizing TRPV3 and TMEM79 Interaction
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HEK293 cells were transfected with mTRPV3 alone or a combination of mTRPV3 and mTMEM79 as described above. After 24 h, cells were fixed in 4% paraformaldehyde (PFA; Wako) for 10 min, permeabilized by PBST (0.25% triton in PBS) for 15 min at RT and blocked with Duolink Blocking Solution (Sigma) for one h at 37 °C. Cells were next incubated with primary mouse anti-TRPV3 (Abcam, ab85022, 1:200) and rabbit anti-TMEM79 (Novus, NBP1-59832, 1:200) antibodies O/N at 4 °C. On the second day, the cells were washed twice with buffer A (0.01 M Tris, 0.15 M NaCl, and 0.05% Tween 20, pH 7.4 adjusted with HCl), and incubated with Duolink PLA Probes anti-mouse PLUS (Sigma, DUO92001) and anti-rabbit MINUS (Sigma, DUO92005) for one h at 37 °C. Thereafter, ligation and amplification were performed with the red Duolink in situ detection reagents kit (Sigma, DUO92008). The plasma membrane was stained with rabbit anti-Na+/K+ ATPase. Cells were then washed twice with buffer B (0.2 M Tris and 0.1 M NaCl, pH 7.5 adjusted with HCl) for 10 min each, rinsed with 0.01% buffer B for 1 min, and mounted with Duolink in situ mounting medium with DAPI (Sigma, DUO82040). Images were obtained using LSM 800 Airyscan (Carl Zeiss).
10
Proximity Ligation Assay for Protein Interactions
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