Otx1 + Otx2 Recombinant Rabbit Monoclonal Antibody [PSH01-62]

☑ Cell treatment (CT)

Western blot analysis of Otx1 + Otx2 on different lysates with Rabbit anti-Otx1 + Otx2 antibody (HA721705) at 1/1,000 dilution.

Lane 1: 293T transfected with FLAG-tagged empty control cell lysate
Lane 2: 293T transfected with FLAG-tagged Otx2 cell lysate

Lysates/proteins at 30 µg/Lane.

Predicted band size: 32 kDa
Observed band size: 32 kDa

Exposure time: 24 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 (HA721705) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.

Immunohistochemical analysis of paraffin-embedded rat eye tissue with Rabbit anti-Otx1 + Otx2 antibody (HA721705) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA721705) at 1/200 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.

☑ Cell treatment (CT)

Immunocytochemistry analysis of 293T overexpress with or without Otx2 cells labeling Otx1 + Otx2 with Rabbit anti-Otx1 + Otx2 antibody (HA721705) at 1/1,000 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-Otx1 + Otx2 antibody (HA721705) at 1/1,000 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.

☑ Cell treatment (CT)

Flow cytometric analysis of 293T overexpress with or without Otx2 cells labeling Otx1 + Otx2.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721705, 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).

Western blot analysis of Otx1 + Otx2 on different lysates with Rabbit anti-Otx1 + Otx2 antibody (HA721705) at 1/1,000 dilution.

Lane 1: Human Otx2 recombinant protein, 50ng/Lane
Lane 2: Human CRX recombinant protein, 50ng/Lane
Lane 3: Human Otx1 recombinant protein, 50ng/Lane

Exposure time: 5 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 (HA721705) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.