Anti-OTX2 antibody [RM1205]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-OTX2 antibody [RM1205] (AB322111)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Y79 (human retinoblastoma cell) cells labelling OTX2 with ab322111 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in Y79 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Negative control : Hela (PMID : 12559959). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Magenta). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human spleen. The section was incubated with ab322111 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-OTX2 antibody [RM1205] (AB322111)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell, Left) / Y79 (human retinoblastoma cell, Right) cells labelling OTX2 with ab322111 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : HeLa (PMID : 12559959).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human liver. The section was incubated with ab322111 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Human retina tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human retina in (A) ab322111 and (B) ab21990. The image on (A) ab322111 is applied with Anti-OTX2 antibody at 1/20000 dilution and (B) ab21990 is applied with Anti-OTX2 antibody at 1/4000 dilution. The section was incubated with ab322111 or ab21990 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on mouse spleen. The section was incubated with ab322111 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on mouse liver. The section was incubated with ab322111 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Mouse choroid plexus tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse choroid plexus in (A) ab322111 and (B) ab21990. The image on (A) ab322111 is applied with Anti-OTX2 antibody at 1/20000 dilution and (B) ab21990 is applied with Anti-OTX2 antibody at 1/4000 dilution. The section was incubated with ab322111 or ab21990 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Mouse retina tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse retina in (A) ab322111 and (B) ab21990. The image on (A) ab322111 is applied with Anti-OTX2 antibody at 1/20000 dilution and (B) ab21990 is applied with Anti-OTX2 antibody at 1/4000 dilution. The section was incubated with ab322111 or ab21990 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OTX2 antibody [RM1205] (AB322111)

Immunohistochemical analysis of paraffin-embedded Mouse E14.5 tissue labeling OTX2 with ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse E14.5. The section was incubated with ab322111 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-OTX2 antibody [RM1205] (AB322111)

OTX2 was immunoprecipitated from 0.35 mg Mouse retina tissue lysate with ab322111 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322111 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse retina tissue lysate

Lane 2 : ab322111 at 1/30 IP in Mouse retina tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322111 in mouse retina tissue lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Immunoprecipitation - Anti-OTX2 antibody [RM1205] (ab322111) at 1/30 dilution

Lane 1:

Mouse retina tissue lysate at 10 µg

Lane 2:

Immunoprecipitation at 10 µg

Secondary

All lanes:

ab322111 at 1/30 IP in Mouse retina tissue lysate at 1/5000 dilution

true

Exposure time: 119s

• WB

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Western blot - Anti-OTX2 antibody [RM1205] (AB322111)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : HeLa (PMID : 12559959)

Anti-OTX2 antibody (ab322111, recombinant monoclonal) showed stronger band than Anti-Otx1 + Otx2 antibody (ab21990, polyclonal) with human samples.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-OTX2 antibody [RM1205] (ab322111) at 1/1000 dilution

Lane 1:

Y79 (human retinoblastoma cell) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,36 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-OTX2 antibody [RM1205] (AB322111)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : spleen

The membrane was incubated with ab322111 at 4℃ overnight.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-OTX2 antibody [RM1205] (ab322111) at 1/1000 dilution

Lane 1:

Mouse retina tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 36 kDa,124 kDa

false

Exposure time: 70s

• WB

Supplier Data

Western blot - Anti-OTX2 antibody [RM1205] (AB322111)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : spleen

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-OTX2 antibody [RM1205] (ab322111) at 1/1000 dilution

Lane 1:

Rat retina tissue lysate at 20 µg

Lane 2:

Rat spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,124 kDa

false

Exposure time: 180s