WB: 1:5,000
IF-Cell: 1:2,000
IF-Tissue: 1:50
IHC-P: 1:50-1:200
IP: Use at an assay dependent concentration.
FC: 1:5,000
Fig1: Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/5,000 dilution and competitor's antibody at 1/5,000 dilution.
Lane 1: MCF7 cell lysate
Lane 2: K-562 cell lysate (negative)
Lane 3: A431 cell lysate
Lane 4: Neuro-2a cell lysate
Lane 5: NIH/3T3 cell lysate
Lane 6: C6 cell lysate
Lane 7: SH-SY5Y cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 34 kDa
Observed band size: 35 kDa
Exposure time: 20 seconds;
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 (ET1601-31) at 1/5,000 dilution and competitor's antibody at 1/5,000 dilution were 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.
Fig2: Cyclin D1 was immunoprecipitated from 0.5 mg Hela whole cell lysates with ET1601-31 at 2 μg/mL. Western blot was performed from the immunoprecipitate using ET1601-31 at 1/500 dilution for 45 minutes at room temperature. Goat anti-Rabbit IgG-HRP Secondary Antibody (HA1001) was used at 1:300,000 dilution for 30 minutes at room temperature.
Lane 1: Hela whole cell lysates at 10 μg;
Lane 2: Cyclin D1 (ET1601-31) IP in Hela whole cell lysates;
Lane 3: Rabbit IgG instead of Cyclin D1 (ET1601-31) in Hela whole cell lysates.
Predicted band size: 34 kDa
Observed band size: 34 kDa
Exposure time: 5 minutes;
12% SDS-PAGE gel.
Fig3: Immunocytochemistry analysis of Neuro-2a cells labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/2,000 dilution and competitor's antibody at 1/1,600 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-Cyclin D1 antibody (ET1601-31) at 1/2,000 dilution and competitor's antibody at 1/1,600 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.
Fig4: Flow cytometric analysis of MCF7 cells labeling Cyclin D1.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-31, red) at 1/5,000 dilution and competitor's antibody (red) at 1/2,000 dilution, 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™ 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).
Fig5: ICC staining of Cyclin D1 in PC-12 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1601-31, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
Fig6: Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Cyclin D1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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.
Fig7: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue using anti-Cyclin D1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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.
Fig8: Immunohistochemical analysis of paraffin-embedded human liver carcinoma tissue using anti-Cyclin D1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-31, 1/50) for 30 minutes 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.
Fig9: Immunohistochemical analysis of paraffin-embedded human small intestine tissue using anti-Cyclin D1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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.
Fig10: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue using anti-Cyclin D1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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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