Cells were plated on 8‐μm‐thick chip, fixed in 4% ice‐cold paraformaldehyde, and permeabilized using 0.5% Triton X‐100/PBS for 20 min at room temperature. The cells were blocked with 10% albumin from bovine serum (Amresco) for 1 h and then incubated with antibodies against vimentin, E‐cadherin (1 : 50; Cell Signaling), DDAH1, and β‐catenin (1 : 50; Abcam) overnight at 4 °C. After three washes, cells were incubated with Alexa Fluor 488‐conjugated goat anti‐rabbit secondary antibody (1 : 200; Invitrogen) or Alexa Fluor 647‐conjugated goat anti‐mouse IgG (1 : 200; Abcam). The nuclei were counterstained with DAPI (Invitrogen), and cells were visualized with a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).
Alexa fluor 647 conjugated goat anti mouse igg
Manufactured by Abcam
Sourced in United Kingdom, United States
Alexa Fluor 647-conjugated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) antibodies. The antibody is conjugated to the Alexa Fluor 647 fluorescent dye, which can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Albumin from bovine serum, by Avantor (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ddah1, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
β catenin, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Abcam (1 mentions)
Mouse igg1 fitc isotype control, by Santa Cruz Biotechnology (1 mentions)
Rabbit igg monoclonal isotype control, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Leagene (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Confocal microscopy, by Olympus (1 mentions)
4 protocols using alexa fluor 647 conjugated goat anti mouse igg
1
Immunofluorescence Staining of Adherent Cells
2
Quantifying GLUT1 Expression in Cells
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Cells were treated
with the various compounds for 1 h and washed with PBS; subsequently,
they were irradiated with light at the indicated wavelength. Next,
the cells were fixed by treating them with PBS containing 4% paraformaldehyde
for 20 min and permeabilized by treating them with 0.1% (v/v) Triton
X-100 for 10 min. After blocking with 2% bovine serum albumin for
30 min, the fixed cells were incubated with mouse anti-GLUT1 antibody
and Alexa Fluor 647-conjugated goat anti-mouse IgG (#ab150115, abcam,
UK) for 1 h. The fluorescence signals were recorded using a confocal
laser microscope (LMS780 spectral confocal system, Zeiss Co., Ltd.,
Germany).
with the various compounds for 1 h and washed with PBS; subsequently,
they were irradiated with light at the indicated wavelength. Next,
the cells were fixed by treating them with PBS containing 4% paraformaldehyde
for 20 min and permeabilized by treating them with 0.1% (v/v) Triton
X-100 for 10 min. After blocking with 2% bovine serum albumin for
30 min, the fixed cells were incubated with mouse anti-GLUT1 antibody
and Alexa Fluor 647-conjugated goat anti-mouse IgG (#ab150115, abcam,
UK) for 1 h. The fluorescence signals were recorded using a confocal
laser microscope (LMS780 spectral confocal system, Zeiss Co., Ltd.,
Germany).
3
Immunofluorescence Analysis of Stem Cell Markers
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Immunofluorescence was performed as previously described10 (link). The following antibodies were used: β-Catenin mouse monoclonal (1:100), YAP1 mouse monoclonal (1:100), Runx2 mouse monoclonal (1:200), Osterix mouse monoclonal (1:500), VEGF-A mouse monoclonal (1:100), mouse IgG1-FITC isotype control (1:200, Santa Cruz, CA, USA), β-Catenin rabbit polyclonal (1:100), CD31 rabbit monoclonal (1:500, Severicebio, Wuhan, China), Rabbit IgG monoclonal isotype control (1:200, Abcam, MA, USA), Alexa Fluor® 647 conjugated Goat Anti-Mouse IgG (1:100), Alexa Fluor® 488 conjugated Goat Anti-Mouse IgG (1:100, Abcam, MA, USA), Cy5 conjugated Goat Anti-rabbit IgG (1:100), Alexa Fluor® 488-conjugated Goat Anti-Rabbit IgG (1:100), Cy3 conjugated Goat Anti-Rabbit IgG (1:100, Severicebio), and DAPI (Leagene, Beijing, China).
4
Proteasomal Regulation of MyD88 by rTcpC
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5×105 cells of K-macrophages from different groups and 4 μg/ml rTcpC treated J774A.1 cells were centrifuged at 800×g for 10 min at 4°C. Mouse anti-MyD88-IgG (Abcam) or rabbit anti-PSMD2-IgG (Abcam) were used as the primary antibody. Alexa Fluor 488-conjugated goat anti-rabbit-IgG and Alexa Fluor 647-conjugated goat anti-mouse-IgG (Abcam) were used as the secondary antibody respectively and DAPI (Sigma) to stain the nucleus. Co-localization of MyD88 with PSMD2 was examined by confocal microscopy (Olympus) (495/519, 652/668 or 485 nm excitation/emission wavelengths for Alexa Fluor 488, Alexa Fluor 647 or DAPI detection). Percentages and yellow fluorescence intensities (FI) reflecting the co-localization were analyzed. Untreated corresponding cells were used as controls. To examine the influence of MG-132 on rTcpC induced accumulation of MyD88 in proteasomes, J774A.1 cells were treated with 1 μM MG-132 for 30 minutes before treatment with 4 μg/ml rTcpC for the indicated time, and then the treated cells are subjected to confocal microscopy as described above.
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