Anti-Otx1 + Otx2 antibody

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (AB21990)

ab21990 staining Otx2 in embryonic day 14.5 (E14.5) mouse eye by Immunohistochemistry (formalin fixed, paraffin embedded sections).
The embryo was fixed in 10% neutral buffered formalin overnight at room temperature, then paraffin-embedded and sectioned at 4μm. Following deparaffinization and antigen retrieval in a rice steamer in 10mM sodium citrate (pH 6.0), the sections were blocked with 5% normal goat serum for half an hour at room temperature. ab21990 was diluted in PBS at 1/100, and incubated overnight at 4°C. The secondary was goat anti-rabbit Alexa^®568, diluted in PBS with 1.5% normal goat serum. The mounting medium contained the nuclear stain DAPI (blue).

This image is courtesy of Erin Bassett

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-Otx1 + Otx2 antibody (AB21990)

The image shows staining of Otx2 in human embryonic stem cells differentiated into neuroectoderm. As would be expected, staining is nuclear.

This image is courtesy of L. Vallier, University of Cambridge

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (AB21990)

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.

• IP

Unknown

Immunoprecipitation - Anti-Otx1 + Otx2 antibody (AB21990)

Otx1 +2 was immunoprecipitated using 0.5mg Y79 whole cell extract, 5μg of Rabbit polyclonal to Otx1 +2 and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Y79 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab21990.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 37kDa; Otx1 +2, non specific - as present in control (lane 2); 37kDa : We are confident this was due to slight lane contamination and the band seen in the IP lane is our target of interest.

All lanes:

Immunoprecipitation - Anti-Otx1 + Otx2 antibody (ab21990)

true

Exposure time: 90s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (AB21990)

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (AB21990)

Mouse E6 embryos were sectioned and then fixed in paraformaldehyde. Antigen retrieval was performed using citric acid and the sections permeablized and blocked for 1 hour in serum. Embryos were stained with ab21990 (1/2500) diluted in 10% serum, 1% ovalbumin in PBSTween for 14 hours at 4°C. They were then washed and stained with a goat anti-rabbit biotin conjugated antibody. After using a biotinylated secondary, ABC and then DAB was used for color development.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (AB21990)

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.

• WB

Unknown

Western blot - Anti-Otx1 + Otx2 antibody (AB21990)

This Otx1 recombinant protein has a GST tag causing it to run at ~74kDa.

All lanes:

Western blot - Anti-Otx1 + Otx2 antibody (ab21990) at 1 µg/mL

All lanes:

OTX1 (Human) - Recombinant Protein at 0.1 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 31.6 kDa

Observed band size: 74 kDa

true

• WB

Supplier Data

Western blot - Anti-Otx1 + Otx2 antibody (AB21990)

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] (<a href='/en-us/products/primary-antibodies/otx2-antibody-rm1205-ab322111'>ab322111</a>) 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

Project

Western blot - Anti-Otx1 + Otx2 antibody (AB21990)

All lanes:

Western blot - Anti-Otx1 + Otx2 antibody (ab21990) at 1 µg/mL

Lane 1:

Y79 Lysate at 20 µg

Lane 2:

Y79 Lysate at 20 µg with Otx2 peptide (<a href='/en-us/products/unavailable/otx2-peptide-ab24347'>ab24347</a>)

Secondary

All lanes:

Alexa Fluor Goat polyclonal to Rabbit IgG at 1/10000 dilution

Predicted band size: 31.6 kDa

Observed band size: 28 kDa,37 kDa

false