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25 ug total protein per lane of various lysates (see on figure) probed with MBP polyclonal antibody, unconjugated (ER1912-82) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.
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Paraformaldehyde-fixed, paraffin embedded Mouse Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
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Paraformaldehyde-fixed, paraffin embedded Mouse Cerebellum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
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Paraformaldehyde-fixed, paraffin embedded Rat Cerebellum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
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Paraformaldehyde-fixed, paraffin embedded Rat Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
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Paraformaldehyde-fixed, paraffin embedded Human Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
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Paraformaldehyde-fixed, paraffin embedded Rat Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4℃. Followed by conjugated Goat Anti-Rabbit IgG antibody (green), DAPI (blue, C02-04002) was used to stain the cell nuclei.
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Paraformaldehyde-fixed, paraffin embedded Human Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4℃. Followed by conjugated Goat Anti-Rabbit IgG antibody (green), DAPI (blue, C02-04002) was used to stain the cell nuclei.
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Paraformaldehyde-fixed, paraffin embedded Mouse Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4℃. Followed by conjugated Goat Anti-Rabbit IgG antibody (Rose red), DAPI (blue, C02-04002) was used to stain the cell nuclei.
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Paraformaldehyde-fixed, paraffin embedded Mouse Cerebellum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4℃. Followed by conjugated Goat Anti-Rabbit IgG antibody (Rose red), DAPI (blue, C02-04002) was used to stain the cell nuclei.
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Paraformaldehyde-fixed, paraffin embedded Rat Cerebellum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4℃. Followed by conjugated Goat Anti-Rabbit IgG antibody (Rose red), DAPI (blue, C02-04002) was used to stain the cell nuclei.
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Paraformaldehyde-fixed, paraffin embedded Human Cerebellum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4℃. Followed by conjugated Goat Anti-Rabbit IgG antibody (Rose red), DAPI (blue, C02-04002) was used to stain the cell nuclei.
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Paraformaldehyde-fixed, paraffin embedded Human Left Parietal Lobe; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with MBP Polyclonal Antibody, Unconjugated (ER1912-82) at 1:200 overnight at 4℃. Followed by conjugated Goat Anti-Rabbit IgG antibody (Rose red), DAPI (blue, C02-04002) was used to stain the cell nuclei.
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Blank control: A549.
Primary Antibody (green line): Rabbit Anti-MBP antibody (ER1912-82)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with PBST for 20 min at room temperature. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
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