NHKs were fixed with 4% paraformaldehyde, permeabilized with methanol, and blocked using an Immunofluorescence application solution kit (Cell Signaling Technology). NHKs were incubated with p65 antibody (1:400 dilution; Cell Signaling Technology) for 2 h and consecutively incubated with Alexa 594 conjugated goat anti-rabbit IgG secondary antibody (1:400 dilution; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h in the dark. NHKs were additionally stained and mounted using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA).
Alexa 594 conjugated goat anti rabbit igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594 conjugated goat anti-rabbit IgG secondary antibody is a detection reagent used to visualize and quantify target proteins in various immunoassays, such as Western blotting, immunohistochemistry, and flow cytometry. It binds to the Fc region of rabbit primary antibodies, allowing for the indirect detection of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview fv1000, by Olympus (3 mentions)
Superblock t20 pbs blocking buffer, by Thermo Fisher Scientific (2 mentions)
Alexa 488 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
P65 antibody, by Cell Signaling Technology (1 mentions)
Immunofluorescence application solution kit, by Cell Signaling Technology (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Superblock pbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd61 monoclonal antibody, by GeneTex (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Wga fitc, by GeneTex (1 mentions)
Alexa 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti ve cadherin monoclonal antibody, by Beckman Coulter (1 mentions)
4 protocols using alexa 594 conjugated goat anti rabbit igg secondary antibody
1
Immunofluorescent Staining of p65 in NHKs
2
Visualizing NS1 Binding on Platelets
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To visualize the binding of NS1 on platelet surfaces, 2×106 washed platelets were plated on 0.01% poly-L-lysine-coated coverslips in 24-well plates and treated with BSA or NS1 (1 μg/ml) at 4°C for 1 h. Subsequently, platelets were fixed with 1% paraformaldehyde with no further permeabilization. After blocking with SuperBlock™ (PBS) Blocking Buffer (Thermo Fisher Scientific) for 1 h, platelets were incubated overnight at 4°C with anti-NS1 monoclonal antibodies (33D2) and anti-CD61 monoclonal antibody (Genetex). After being washed with PBS, platelets were incubated with Alexa 488-conjugated goat anti-mouse IgG secondary antibody and Alexa 594-conjugated goat anti-rabbit IgG secondary antibody (1:500; Thermo Fisher Scientific) for 1 h. The coverslips were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA). The images were acquired using a confocal microscope (Olympus FluoView FV1000).
3
Visualizing Endothelial Glycocalyx Integrity
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HUVECs were seeded as a monolayer onto a microscope cover glass slide and cultured under different conditions. After treatment for indicated time, the cells were fixed in 2% paraformaldehyde and then blocked with Superblock T20 (PBS) blocking buffer (Thermo Fisher Scientific).To measure the integrity of the endothelial glycocalyx and the deposition of CD138, the expression of sialic acid was stained with wheat germ agglutinin (WGA) lectin conjugated to FITC (WGA-FITC, Genetex) and the distribution of HPA-1 and CD138 was detected by anti-mouse-CD138 mAb (BD, Franklin Lakes, NJ, USA) or rabbit anti-HPA-1 polyclonal antibody (GeneTex). Primary antibodies were incubated with the fixed monolayer overnight at 4°C, followed by incubation with Alexa 488-conjugated goat anti-mouse IgG secondary antibody, Alexa 594-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, CA, USA) (1:500 diluted) and Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) (1:3,000 diluted) for 1 h. Images were captured using a confocal microscope (Olympus FluoView FV1000, Melville, NY, USA).
4
Immunofluorescence Analysis of Cell-Cell Junctions and Signaling Receptors
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A monolayer of cells was seeded onto a microscope cover glass slide. After 30 min of treatment, the cells were fixed in 4% paraformaldehyde for 15 min followed by three washes with PBS. The cells were subsequently blocked with Superblock T20 (PBS) Blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. To detect VE-cadherin, LC3 puncta localization, or the expression of the MIF receptors, a mouse anti-VE-cadherin monoclonal antibody (Beckman Coulter), a rabbit anti-LC3 polyclonal antibody (GeneTex), a goat anti-CD74 polyclonal antibody (SantaCruz), a rabbit anti-CXCR4 polyclonal antibody (GeneTex), or a rabbit anti-CXCR7 (GeneTex) polyclonal antibody (1:200 diluted in PBS) were incubated with the cells overnight at 4°C. After three washes with Tris-buffered saline-Tween 20 (TBST), the cells were incubated with an Alexa 488-conjugated goat anti-mouse IgG secondary antibody, an Alexa 594-conjugated goat anti-rabbit IgG secondary antibody or an Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, Calif) (1:500 diluted) and Hoechst 33342 (Invitrogen) (1:3,000 diluted) for 1 h, and the slides were washed 3 times with PBS. The images were acquired using a confocal microscope (Olympus FluoView FV1000, Melville, NY).
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