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☑ Relative expression (RE)
Western blot analysis of NCAM1 on different lysates with Rabbit anti-NCAM1 antibody (HA722756) at 1/3,000 dilution.
Lane 1: SH-SY5Y cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (negative) (20 µg/Lane)
Lane 3: C6 cell lysate (20 µg/Lane)
Lane 4: Rat brain tissue lysate (20 µg/Lane)
Predicted band size: 95 kDa
Observed band size: 120-250 kDa
Exposure time: 5 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722756) at 1/3,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
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Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-NCAM1 antibody (HA722756) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA722756) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-NCAM1 antibody (HA722756) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA722756) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunocytochemistry analysis of SH-SY5Y cells labeling NCAM1 with Rabbit anti-NCAM1 antibody (HA722756) at 1/1,000 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-NCAM1 antibody (HA722756) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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Immunocytochemistry analysis of C6 cells labeling NCAM1 with Rabbit anti-NCAM1 antibody (HA722756) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-NCAM1 antibody (HA722756) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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Flow cytometric analysis of HeLa (left, negative) and SH-SY5Y (right, positive) cells labeling NCAM1.
Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA722756, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 594 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1122) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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Flow cytometric analysis of human peripheral blood lymphocytes labeling NCAM1.
Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA722756, 1μg/mL) (red). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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NCAM1 was immunoprecipitated from 0.2 mg SH-SY5Y cell lysate with HA722756 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA722756 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: SH-SY5Y cell lysate (input)
Lane 2: HA722756 IP in SH-SY5Y cell lysate
Lane 3: Rabbit IgG instead of HA722756 in SH-SY5Y cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 2 seconds; ECL: K1801
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