Anti-Oct4 antibody [EPR17980]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17980] (AB200834)

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling Oct4 with ab200834 at 1/500 dilution, followed Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Human cerebral cortex tissue is a negative control for Oct4. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17980] (AB200834)

Immunohistochemical analysis of paraffin-embedded Human spermatocytoma tissue labeling Oct4 with ab200834 at 1/500 dilution, followed Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and weakly cytoplasmic staining on Human spermatocytoma tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17980] (AB200834)

Immunohistochemical analysis of paraffin-embedded Human dysgerminoma of ovary tissue labeling Oct4 with ab200834 at 1/500 dilution, followed Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and weakly cytoplasmic staining on Human dysgerminoma of ovary tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ChIP-seq

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ChIP-sequencing - Anti-Oct4 antibody [EPR17980] (AB200834)

Chromatin was prepared from NCCIT (Human pluripotent embryonic carcinoma cell line) cells. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab200834 [EPR17980]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody [EPR17980] (AB200834)

ab200834 staining Oct4 in F9 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab200834 at a 5μg/ml concentration and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1μg/ml concentration, followed by a further incubation at room temperature for 1h with an anti-rabbit AlexaFluor® 488 (ab150081) at 2 μg/ml (shown in green) and an anti-mouse AlexaFluor® 594 (ab150120) at 2 μg/ml (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Oct4 antibody [EPR17980] (AB200834)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed F9 (Mouse embyro testicular cancer cell line) cells labeling Oct4 with ab200834 at 1/60 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730;black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody [EPR17980] (AB200834)

Immunocytochemistry/Immunofluorescence analysis of F9 (mouse embryonal carcinoma) labelling Oct4 with purified ab200834 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).

Control : PBS only

• IP

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Immunoprecipitation - Anti-Oct4 antibody [EPR17980] (AB200834)

Oct4 was immunoprecipitated from 1mg of F9 (Mouse embyro testicular cancer cell line) whole cell lysate with ab200834 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab200834 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : F9 whole cell lysate 10 μg (Input). Lane 2 : ab200834 IP in F9 whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab200834 in F9 whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 5 seconds

All lanes:

Immunoprecipitation - Anti-Oct4 antibody [EPR17980] (ab200834)

Predicted band size: 38 kDa

false

• WB

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Western blot - Anti-Oct4 antibody [EPR17980] (AB200834)

Low levels of expression in adult tissues

Blocking/Dilution Buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Oct4 antibody [EPR17980] (ab200834) at 1/1000 dilution

Lane 1:

Human placenta lysate at 10 µg

Lane 2:

Human fetal brain lysate at 10 µg

Lane 3:

Human testis lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 38 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Oct4 antibody [EPR17980] (AB200834)

Low levels of expression in adult tissues.

Blocking/Dilution Buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Oct4 antibody [EPR17980] (ab200834) at 1/10000 dilution

Lane 1:

NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate at 10 µg

Lane 2:

NTera-2/D1 (Human malignant pluripotent embryonic carcinoma) whole cell lysate at 10 µg

Lane 3:

F9 (Mouse embyro testicular cancer cell line) whole cell lysate at 10 µg

Lane 4:

JAR (Human placenta choriocarcinoma cell line) whole cell lysate at 10 µg

Lane 5:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 6:

Mouse testis lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 38 kDa

Observed band size: 45 kDa

false

Exposure time: 30s

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-Oct4 antibody [EPR17980] (AB200834)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 NCCIT (Human pluripotent embryonic carcinoma cell line) cells and 5 µg of ab200834 [EPR17980]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The ChIP data was conducted on chromatin prepared from NCCIT cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab200834. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.