Cells were treated for 24 hours with TNFα (200 ng/mL), or PTHrP(12-48) (50 nM) on day 0 and day 7. Cells were fixed, permeabilized, and stained using a specific primary cleaved caspase 3 (Asp175) antibody(30 ) at 1:400 dilution in PBS. Cells were washed in PBS three times followed by incubation for 1 hour with secondary antibodies (Supplemental Table 1 ). For both assays, ProLong Gold Antifade reagent with 4,6-diamidino-2-phenylindole (DAPI; Cell Signaling, Danvers, MA, USA), used to stain nuclei (blue), was added. Coverslipped slides were allowed to dry in the dark at RT overnight and then sealed using clear nail polish and kept at 4°C. To visualize cells, either fluorescence microscopy was performed (Evos-FL microscope; Advanced Microscopy group [AMG], Bothell, WA, USA), fitted with light cubes for GFP, DAPI, Texas Red, and red fluorescent protein [RFP]), or confocal fluorescent microscopy was performed (Zeiss LSM 510 confocal microscope; Zeiss, Oberkochen, Germany). Images obtained using DAPI (405 nm), FITC/GFP (488 nm), and Cy5 (561 nm) light cubes were processed using Zen 2009 software (Zeiss, Oberkochen, Germany).
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi
Manufactured by Cell Signaling Technology
Sourced in United States
Prolong Gold AntiFade reagent with 4',6-diamidino-2-phenylindole (DAPI) is a mounting medium designed for fluorescence microscopy. The product contains DAPI, a blue-fluorescent DNA-binding dye that selectively stains nuclei. The reagent helps preserve fluorescent signals and prevent photobleaching.
Automatically generated - may contain errors
Lab products found in correlation
Airyscan confocal microscope, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Zen 2009, by Zeiss (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Thunder imager, by Leica (1 mentions)
Mica microscope, by Leica (1 mentions)
Saponin, by Merck Group (1 mentions)
Vegfr2, by Abcam (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Ab15148, by Abcam (1 mentions)
Alexa fluor 555 conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ab92547, by Abcam (1 mentions)
9 protocols using prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi
1
Cleaved Caspase-3 Immunofluorescence Assay
2
Visualizing Autophagy Induction in Cells
3
Quantification of C5b-9 in HBV-transfected Huh7 cells
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HBV transfected Huh7 cells were grown as monolayer on poly-l -lysine coated coverslips in 6-well plate for 48 h and then treated with 50 μl sera from HC or CHB patients or with CHB patient sera supplemented with recombinant C9 protein at final concentration of 15 ng/ml of culture media for 60 min at 37 °C. The cells were fixed with 4% paraformaldehyde for 10 min and after washing with ice cold TBS, the masked antigens were retrieved by treatment with 0.05% trypsin-calcium chloride solution [trypsin 0.5%, calcium chloride 1% (pH 7.8)] at 37 °C for 20 min. After washing with TBS containing 0.025% TritonX-100, blocking was done with 1% BSA in TBS for 2 h. The cells were stained overnight at 4 °C with 1:500 dilution of mouse anti human anti-C5b-9 primary antibody (Santa Cruz Biotechnology) followed by incubation with 1:1000 dilution of goat anti-mouse IgG secondary antibody conjugated to Alexa fluor 488 (Thermo Fisher Scientific) for 1 h at 37 °C. Pro-Long Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology) was used for nuclear counterstaining and mounting. Images were captured in Thunder imager (Leica) at 40× magnification.
4
Immunofluorescence Microscopy of S. flexneri
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S. flexneri 2a were grown as described above, and a 100 µL suspension (107 CFU/mL) was added to microscope chamber slides. Bacteria were fixed in aqueous 4% paraformaldehyde for 45 min at RT and then washed with PBS. After washing, fixed bacteria were permeabilized and blocked for 1 h at with PBS containing 15% FBS, 2% BSA, and 0.1% Saponin (all from Sigma-Aldrich), then rinsed with PBS and incubated overnight at 4 °C with pooled sera from mice immunized IM with VirGα or AdjuPhos® diluted 1:100 in PBS containing 15% FBS and 0.2% BSA. Stained bacteria were washed twice with PBS and incubated with AF555-labeled goat anti-mouse IgG (Catalogue # A32727, Thermo Fisher Scientific) diluted 1:100 in PBS for 1 h at RT in the dark. After washing, bacteria were mounted in ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology, Danvers, MA) for DNA staining at 4 °C for at least 24 h. Confocal images were obtained using a MICA microscope (Leica, Wetzlar Germany). Images were captured with a 60× water immersion objective, and settings were adjusted to optimize the signal. Images were collated using FIJI/ImageJ (NIH). Signal processing was applied equally across the entire image.
5
Immunofluorescence Assay for NF-κB Activation
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The immunofluorescence (IF) experiment was performed as described previously [4 (link), 19 (link)]. The MH-S cells were rinsed with DPBS and fixed for 10 min in 4% paraformaldehyde. The cells were also permeabilized for 10 min with 0.2% triton X-100 in TBS (TBST) and rinsed three times with TBST for 5 min each time. The samples were blocked for 1 h with 2% BSA before adding rabbit anti-p-NF-κB (primary antibody) overnight at 4°C. MH-S cells were rinsed three times with TBST for 5 min each time. The samples were incubated for 1 h in the dark with a secondary antibody and then washed three times with TBST before being mounted with a Prolong Gold Antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology) to observe the nuclei and examined using confocal microscopy (LSM700, Carl Zeiss, Jena, Germany).
6
Immunostaining of Endothelial and Smooth Muscle Cells
7
Immunofluorescence Analysis of CD34 and VEGFR2
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Sections were incubated with a primary antibody to CD34 (dilution 1 : 100; Abcam) and vascular endothelial growth factor receptor 2 (VEGFR2) (dilution 1 : 100; Abcam) overnight at 4°C. The sections were then incubated with appropriate secondary antibodies (Alexa Fluor™ Plus 488 donkey anti-rabbit IgG (1 : 25) and Alexa Fluor™ Plus 555 donkey anti-mouse IgG (1 : 25); Thermo Fischer Scientific) for 2 hours at RT. Nuclei were counterstained with ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology, Danvers, MA, USA) and DAPI (Thermo Fischer Scientific). A Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) and Leica Application Suite X v.3.4.2.18368 image-processing software (Leica Microsystems) were used for image acquisition and analysis.
8
Apoptosis Detection in Tissue Sections
9
Immunofluorescence analysis of E-cadherin and Vimentin
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AGS cells with or without knockdown of EMP3 were grown on coverslips. After overnight incubation, the cells were fixed, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA). The coverslips were incubated with anti-E-cadherin (ab15148; Abcam) or anti-Vimentin (ab92547; Abcam) at 4°C for overnight. After washing, the coverslips were further incubated with anti-rabbit Alexa Fluor ® 488 conjugated secondary antibody (#4412; Cell Signaling Technology, Danvers, MA, USA) or anti-rabbit Alexa Fluor ® 555 conjugated secondary antibody (#4413; Cell Signaling Technology) for 1 h at room temperature in the dark. Nuclei were stained using Prolong ® Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI; #8961; Cell Signaling Technology). Immunofluorescent images were obtained using an Olympus IX73 inverse microscope (Tokyo, Japan).
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