H9c2 cells were fixed with 3.7% formaldehyde (Sigma) for 15 min, permeabilized with 0.5% Triton X-100/PBS for 10 min, blocked with blocking buffer (5% goat serum, 0.2% Tween-20 in PBS) for 20 min and incubated for 1 h with anti Myc-Tag (9B11) antibody (Cell Signaling) diluted 1:2000 in blocking buffer. All steps were carried out at room temperature. Either ALEXA 488- or ALEXA 594-conjugated antibodies (1:200, Molecular Probes) were used to detect immune complexes. 4′,6′-diamidino-2-phenylindole (DAPI, Sigma) (0.5 mg/mL PBS) was used to visualize DNA. For immunohistochemical analysis, the slides were deparaffinized and rehydrated. Endogenous peroxidase was blocked by immersing tissue sections in methanol containing 3% H2O2, followed by washing in PBS. Non-specific antigens were blocked by incubation of the tissue sections with 2.5% (v/v) horse serum for 20 min. The sections were then incubated for overnight at 4°C with rabbit polyclonal anti-Bax antibody (Santa Cruz). After extended washing in PBS, primary antibody staining was visualized using the VECTASTAIN ABC Kit with Nova RED (Vector Laboratories). Tissue sections were counterstained with haematoxylin, dehydrated and mounted.
Anti myc tag 9b11 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti Myc-Tag (9B11) antibody is a monoclonal antibody that recognizes the Myc epitope tag. This antibody can be used to detect and monitor the expression of proteins tagged with the Myc epitope.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa 594 conjugated antibodies, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Anti β tubulin antibody, by Cell Signaling Technology (1 mentions)
Anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
App y188, by Abcam (1 mentions)
Anti 6e10 antibody, by BioLegend (1 mentions)
Anti cd3 145 2c11, by BD (1 mentions)
Cba flex set, by BD (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Lsm 800 confocal laser scanning microscopy, by Zeiss (1 mentions)
Zen blue edition software, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
4 protocols using anti myc tag 9b11 antibody
1
Immunofluorescence and Immunohistochemistry Protocols
2
Antibody Characterization for Cell Biology
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The following antibodies were used: anti-Myc-Tag (9B11) antibody (Cell Signaling, Danvers, MA, USA), anti-HA-Tag (6E2) antibody (Cell Signaling, Danvers, MA, USA), anti-V5 antibody (Invitrogen, Waltham, MA, USA), anti-β-tubulin antibody (Cell Signaling, Danvers, MA, USA), APP Y188 (Abcam, Cambridge, UK), anti-6E10 antibody (BioLegend, San Diego, CA, USA).
3
Quantifying Activated CAR.OTI T Cell Cytokines
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Activated CAR.OTI T cells (105) were cultured in 96-well flat-bottom plates in T cell media, precoated overnight with PBS, 1 μg mouse IgG2a Isotype control (Cell Signaling) (clone7H8/50), 1 μg/mL anti-CD3 (145-2C11) (BD Biosciences), or 1 μg/mL anti-myc-Tag antibody (9B11; Cell Signaling Technology) for 5 h. Cytokine production was measured from 10 μL of culture supernatant of CTLs using CBA flex sets (BD Biosciences) for mouse IFN-γ and TNF, according to the manufacturer’s instructions. Samples were analyzed on a FACS VERSE using FCAP Array software version 3.0 (Softflow) and the concentration of cytokine was determined to a standard curve and plotted as picograms per milliliter of cytokine.
4
Glucocorticoid Receptor Translocation Assay
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HT-22 cells on poly-D-lysine coated coverslips were transfected with GFP-GR and Myc-GR using Lipofectamine 3000 reagent. After 24 h, culture media was exchanged with serum-free DMEM/High Glucose and then incubated at 37°C for 12 h for serum starvation. After treatment with 10μM dexamethasone and 5μM PDE5 inhibitors for 6 h, the cells were fixed in 4% paraformaldehyde in PBS for 10 min and then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. The fixed HT-22 cells were blocked with 10% normal goat serum (Abcam) for 1 h at room temperature and incubated at 4 °C overnight with anti-Myc tag antibody (9B11) (Cell Signaling Technology). After washing with PBS, the cells were incubated with goat anti-mouse lgG H&L Alexa 647 (Abcam) at room temperature for 1 h and then washed with PBS. Coverslips were mounted with mounting medium with DAPI (Vector Laboratories). Fluorescence confocal images were obtained using LSM 800 confocal laser scanning microscopy (Zeiss). DAPI was used for nuclear staining. Mean fluorescence intensity in the nuclei and whole cells was measured using Zen Blue Edition software (Zeiss).
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