Cytokeratin 7 Recombinant Rabbit Monoclonal Antibody [ST50-05]

☑ Knockdown (KD)

All lanes: Western blot analysis of Cytokeratin 7 with anti-Cytokeratin 7 antibody [ST50-05] (ET1609-62) at 1:1,000 dilution.
Lane 1/2: Wild-type Hela whole cell lysate (20 µg).
Lane 3/4: Cytokeratin 7 fragment 1 knockdown Hela whole cell lysate (20 µg).
Lane 5/6: Cytokeratin 7 fragment 2 knockdown Hela whole cell lysate (20 µg).

ET1609-62 was shown to specifically react with Cytokeratin 7 in wild-type Hela cells. Weakened bands were observed when Cytokeratin 7 knockdown samples were tested. Wild-type and Cytokeratin 7 knockdown samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1609-62, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH , ET1601-4, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

☑ Relative expression (RE)

Western blot analysis of Cytokeratin 7 on different lysates with Rabbit anti-Cytokeratin 7 antibody (ET1609-62) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: A549 cell lysate
Lane 3: MCF7 cell lysate (negative)

Lysates/proteins at 15 µg/Lane.

Predicted band size: 51 kDa
Observed band size: 55 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 (ET1609-62) at 1/5,000 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.

Immunocytochemistry analysis of A549 cells labeling Cytokeratin 7 with Rabbit anti-Cytokeratin 7 antibody (ET1609-62) 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-Cytokeratin 7 antibody (ET1609-62) 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.

Immunohistochemical analysis of paraffin-embedded human breast tissue with Rabbit anti-Cytokeratin 7 antibody (ET1609-62) at 1/1,500 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 (ET1609-62) at 1/1,500 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.

Immunohistochemical analysis of paraffin-embedded human breast tissue with Rabbit anti-Cytokeratin 7 antibody (ET1609-62) at 1/1,500 dilution.

The section was not undergone antigen retrieval. 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 (ET1609-62) at 1/1,500 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.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue with Rabbit anti-Cytokeratin 7 antibody (ET1609-62) at 1/400 dilution.

The section was not undergone antigen retrieval. 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 (ET1609-62) at 1/400 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.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue with Rabbit anti-Cytokeratin 7 antibody (ET1609-62) at 1/1,500 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 (ET1609-62) at 1/1,500 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.

Immunocytochemistry analysis of SK-Br-3 cells labeling Cytokeratin 7.
Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes and blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with the primary antibodies Cytokeratin 7 (ET1609-62, red) at 1/100 dilution for overnight at 4 ℃. Goat anti rabbit IgG (iFluorTM 594) (HA1122) was used as the secondary antibody at 1/1,000 dilution dilution. DAPI was used as nuclear counterstain.

Immunofluorescence analysis of paraffin-embedded human breast tissue labeling Cytokeratin 7 (ET1609-62).

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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibodies Cytokeratin 7 (ET1609-62, red) at 1/400 dilution at +4℃ overnight, washed with PBS.

Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) was used as the secondary antibodies at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Immunofluorescence analysis of paraffin-embedded human liver tissue labeling Cytokeratin 7 (ET1609-62).

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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibodies Cytokeratin 7 (ET1609-62, red) at 1/400 dilution at +4℃ overnight, washed with PBS.

Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) was used as the secondary antibodies at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Flow cytometric analysis of A549 cells labeling Cytokeratin 7.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1609-62, 1μg/mL) (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™ 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).

mIHC analysis of human gastric cancer tissue (Formalin/PFA-fixed paraffin-embedded sections) with Rabbit anti-Cytokeratin 7 antibody (ET1609-62) at 1/1,000 dilution. The immunostaining was performed with the IRISKit® HyperView mTSA Kit (MH900206). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.