Inflorescences from pA9::RDR2-Flag rdr2 plants were embedded in paraffin and sectioned transversely as previously described 33 . The dewaxed and rehydrated sections 33 were treated with 1× PBS containing 100 µg/ml proteinase K for 10 minutes at room temperature, followed by 3 times of wash in 1× PBS. Next, the sections were incubated in 1× PBS containing 2.5% horse serum (Thermo Fisher Scientific, cat. no. 16050130) for 2h at room temperature and then rinsed with 1× PBS for 15 min. Subsequently, the sections were incubated in 1× PBS containing 1% (1:100 dilution) mouse monoclonal anti-Flag M2 primary antibody (Merck, cat. no. F1804) and 0.1% (w/v) BSA for 3-4h. Slides were then washed in 1× PBS twice for 10 minutes each and incubated for 2h with a goat anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific, cat. no. A-21424) secondary antibody (1:500 dilution), followed by a wash for 10 min in 1× PBS. Finally, the sections were mounted under a coverslip with p-phenylenediamine anti-fade mounting agent 85 and examined under a DM6000 microscope (Zeiss).
Mouse monoclonal anti flag m2 primary antibody
Manufactured by Merck Group
Sourced in Spain
The Mouse monoclonal anti-Flag M2 primary antibody is a laboratory reagent used to detect and capture proteins tagged with the Flag epitope. It is a mouse-derived monoclonal antibody that specifically binds to the Flag peptide sequence.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Dm6000 microscope, by Zeiss (2 mentions)
4 6 diamino 2 phenylindole dapi, by Thermo Fisher Scientific (2 mentions)
Laser scanning confocal microscope, by Nikon (2 mentions)
Dylight 488 fluorescence labeled secondary goat anti mouse antibody, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Scharlab (1 mentions)
4 protocols using mouse monoclonal anti flag m2 primary antibody
1
Immunolocalization of RDR2-FLAG Protein
2
Immunolocalization of RDR2-FLAG Protein
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Inflorescences from pA9::RDR2-Flag rdr2 plants were embedded in paraffin and sectioned transversely as previously described 33 . The dewaxed and rehydrated sections 33 were treated with 1× PBS containing 100 µg/ml proteinase K for 10 minutes at room temperature, followed by 3 times of wash in 1× PBS. Next, the sections were incubated in 1× PBS containing 2.5% horse serum (Thermo Fisher Scientific, cat. no. 16050130) for 2h at room temperature and then rinsed with 1× PBS for 15 min. Subsequently, the sections were incubated in 1× PBS containing 1% (1:100 dilution) mouse monoclonal anti-Flag M2 primary antibody (Merck, cat. no. F1804) and 0.1% (w/v) BSA for 3-4h. Slides were then washed in 1× PBS twice for 10 minutes each and incubated for 2h with a goat anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific, cat. no. A-21424) secondary antibody (1:500 dilution), followed by a wash for 10 min in 1× PBS. Finally, the sections were mounted under a coverslip with p-phenylenediamine anti-fade mounting agent 85 and examined under a DM6000 microscope (Zeiss).
3
Immunofluorescence Imaging of Transfected SOX10
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NIH3T3 cells were grown on 6-well plates with cover-slides and transfected with pCMV-3xFlag-SOX10 (wild-type SOX10) or SOX10 variant expression plasmids, following an established protocol (Zhang et al., 2012 (link)). At 48 h after transfection, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde at room temperature for 30 min, and permeabilized in PBS plus 0.2% Triton X-100 (Scharlau, Spain) for 1 h. The reaction was then stopped with blocking solution (PBS, 3% bovine serum albumin plus 5% goat serum) at room temperature for 1 h, and the slides were stained with mouse monoclonal anti-Flag M2 primary antibody (1:600 dilution; Sigma) at 4°C overnight, washed three times with PBS plus 0.1% Triton X-100, and then incubated for 2 h with DyLight 488 fluorescence-labeled secondary goat anti-mouse antibody (1:300 dilution; Thermo Fisher Scientific). The cells were then incubated with 4′,6′-diamino-2-phenylindole (DAPI, Invitrogen) for 3 min before immunofluorescence analysis using a laser scanning confocal microscope (Nikon, Japan) and the NIS-Elements Viewer software.
4
Immunofluorescence Imaging of Flag-tagged MITF
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NIH3T3 cells were transfected with 200 ng of pCMV10 3X Flag wild-type MITF or MITF variant expression plasmids following an established protocol15 (link). At 48 h after transfection, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized in PBS plus 0.2% Triton X-100 (Scharlau, Spain) for 1 h, blocked with blocking solution (PBS, 3% bovine serum albumin plus 5% goat serum) at room temperature for 1 h, stained with mouse monoclonal anti-Flag M2 primary antibody (1:600 dilution, Sigma) at 4 °C overnight, washed with PBS for three times, and then incubated for 2 h with DyLight 488 fluorescence-labeled secondary goat anti-mouse antibody (1:300 dilution, Thermo Fisher Scientific). The cells were incubated with 4,6-diamino-2-phenylindole (DAPI, Invitrogen) for 3 min before immunofluorescence analysis using a laser scanning confocal microscope (Nikon, Japan) and the NIS-Elements Viewer software package.
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