The transfectants were plated on chamber slides (Becton-Dickinson Falcon, Franklin Lakes, NJ, USA) at 50% confluency, washed with ice-cold PBS, fixed with 4% paraformaldehyde-PBS for 20 min, and then permeabilized in PBS containing 0.2% Triton X-100 as described previously 24 . Fixed cells were incubated with a blocking solution containing 0.5% bovine serum albumin for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C. We used the following primary antibodies: rabbit anti-ARNT2 polyclonal antibody (Santa Cruz Biotechnology) and mouse anti-GLUT-1 monoclonal antibody (Arigo Biolaboratories). After washing with PBS, the cells were incubated with secondary antibodies for 1 hour at room temperature. The secondary antibodies were fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA) or Texas Red-conjugated anti-mouse IgG antibody (Vector Laboratories) incubated for 1 hour at room temperature in the dark. Finally, the sections were washed three times with PBS and mounted using Mounting Medium with DAPI (Vector Laboratories). The immunofluorescence was performed by confocal microscopy and analyzed using FluoView Software (Olympus Optical, Tokyo, Japan).
Texas red conjugated anti mouse igg antibody
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
The Texas Red-conjugated anti-mouse IgG antibody is a secondary antibody that can be used to detect and visualize mouse primary antibodies in various immunochemical applications. The antibody is conjugated with the Texas Red fluorescent dye, which can be detected using appropriate fluorescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview software, by Olympus (1 mentions)
Mounting medium with dapi, by Vector Laboratories (1 mentions)
Rabbit anti arnt2 polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Anti s100β, by Agilent Technologies (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Mouse anti βiii tubulin antibody, by Promega (1 mentions)
2 protocols using texas red conjugated anti mouse igg antibody
1
Immunofluorescence Staining of Transfectants
2
Visualizing Neurite Outgrowth and Schwann Cell Phenotype
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NG108-15 neuronal cells, and primary Schwann cells, were immunolabelled for specific proteins to visualize neurite outgrowth from neuronal cells and confirm Schwann cell phenotype, respectively, as previously described [24 (link), 27 ]. Briefly, samples were fixed with 3.7% (v/v) paraformaldehyde (20 min), permeabilized with 0.1% Triton X-100 (20 min) and blocked with 3% bovine serum albumin (BSA), in PBS (30 min) [24 (link), 27 ].
NG108-15 neuronal cells were labelled with a mouse anti-β III-tubulin antibody (1:250) (Promega, UK), for 48 h at 4°C, followed by a Texas Red-conjugated anti-mouse IgG antibody (1:200 dilution in 1% BSA from Vector Labs, USA), for 90 min at room temperature, to visualize and measure neurite outgrowth [24 (link), 27 ].
Schwann cells were identified with a polyclonal rabbit anti-S100β (1:250) (Dako, Denmark) antibody, incubated for 48 h at 4°C, followed by a FITC-conjugated secondary anti-rabbit IgG antibody (1:100 dilution in 1% BSA), for 90 min at room temperature [24 (link), 27 ]. All cells were incubated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich) (300 nM) for 30 min, at room temperature to observe cell nuclei [27 ]. Cells, and samples, were imaged using an upright Zeiss LSM 510 confocal microscope using three fields of view for quantitative analysis [24 (link), 27 ].
NG108-15 neuronal cells were labelled with a mouse anti-β III-tubulin antibody (1:250) (Promega, UK), for 48 h at 4°C, followed by a Texas Red-conjugated anti-mouse IgG antibody (1:200 dilution in 1% BSA from Vector Labs, USA), for 90 min at room temperature, to visualize and measure neurite outgrowth [24 (link), 27 ].
Schwann cells were identified with a polyclonal rabbit anti-S100β (1:250) (Dako, Denmark) antibody, incubated for 48 h at 4°C, followed by a FITC-conjugated secondary anti-rabbit IgG antibody (1:100 dilution in 1% BSA), for 90 min at room temperature [24 (link), 27 ]. All cells were incubated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich) (300 nM) for 30 min, at room temperature to observe cell nuclei [27 ]. Cells, and samples, were imaged using an upright Zeiss LSM 510 confocal microscope using three fields of view for quantitative analysis [24 (link), 27 ].
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