Immunofluorescence and confocal microscopy were performed as previously described [21 (link)]. For mitotic aberrations, cells were stained with α/γ-tubulin primary antibodies (Supplementary Table 1 ) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) mounting media (Vector Laboratories). Images were obtained on a Zeiss AxioObserver LSM780 with objective alpha plan-apochromat 100×/1.46 oil iris.
Axioobserver lsm780
Manufactured by Zeiss
Sourced in Germany
The AxioObserver LSM780 is a laser scanning confocal microscope system manufactured by Zeiss. It is designed for high-resolution imaging and analysis of biological samples. The system utilizes a laser as the excitation source and a sensitive detector to capture detailed images of fluorescently labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole dapi mounting media, by Vector Laboratories (1 mentions)
Alexa fluor 633 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 dye, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti rabbit igg secondary antibody, by Abcam (1 mentions)
3 protocols using axioobserver lsm780
1
Immunofluorescence Analysis of Mitotic Aberrations
2
Visualizing VEGFR2 and αvβ3 Colocalization
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HUVECs (5 × 104 cells/well) were plated in glass coverslips, previously coated with fibronectin (1 μg/cm2), in serum-supplemented DMEM and left overnight in an incubator at 37 °C, 5% CO2. DisBa-01 (1000 nM), previously labelled using Alexa Fluor® 546 dye (Invitrogen, Thermo Scientific), was added to the cells for 2 min. Samples were fixed in 4% paraformaldehyde for 10 min and permeabilized using 0.5% Triton X-100 for 10 min. Samples were washed with PBS, followed by a 1-h incubation in 5% PBS-BSA to block unspecific sites. Cells were incubated overnight with targeted primary antibodies (1:100 Rabbit pAb to VEGF Receptor 2; 1:100 Mouse Monoclonal to the integrin αvβ3, Abcam). Then, secondary antibodies (1:1000 Alexa Fluor 633 goat anti-rabbit, ThermoFisher; 1:1000 Goat polyclonal anti-mouse Alexa Fluor 488, ThermoFisher Scientific) were mixed in 5% PBS-BSA and applied on the wells. After incubation, slides were cleaned and samples were stained with DAPI (Thermo Fisher Scientific) for 10 min. Slides were assembled using ProLong™ Antifade Reagents for Fixed Cells (Thermo Fisher Scientific) and observed on confocal microscope Axio Observer LSM 780 (Zeiss) aided by ZEN BLACK software. Analysis occurred under the same laser intensity for different fluorescences at 63x magnification. Colocalization coefficients were determined using ImageJ FIJI program.
3
NF-κB p65 Nuclear Translocation Assay
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Nuclear translocation of the NF-κB p65 subunit in HDVSMCs was determined by using immunofluorescence staining. HDVSMCs were cultivated on a Millicell EZ Slide 4-well glass slide box and stimulated with Tp0136 at a concentration of 10 μg/mL for 1 h. Thereafter, cells were fixed and permeabilized for 15 min at room temperature with Triton X-100 (0.25%) in 4% formaldehyde. Then the slides with primary antibodies against NF-κB p65 (Cell Signaling Technology, Inc., MA, USA) were incubated at a dilution of 1:1000 overnight at 4 °C overnight, after that, cells were incubated for 2 h at room temperature with a FITC-conjugated anti-rabbit IgG secondary antibody (Abcam, Cambridge, USA) at a dilution of 1:800. Finally, the slides were treated with 4’,6-Diamidino-2-phenylindole (DAPI) for nuclear staining, and the translocation of p65 into the nucleus was observed using a confocal microscope (Zeiss Axio Observer LSM780, Oberkochen, Germany). HDVSMCs were treated with 10 μg/mL Tp0136 for 1 h after preincubating with LY294002, SP600125, SB203580, or BAY11-7082 for 1 h for the inhibition assay. Lastly, the analysis of NF-κB p65 nuclear translocation was performed as previously mentioned.
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