Anti-Digoxigenin antibody [EPR26829-63]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Digoxigenin antibody [EPR26829-63] (AB320717)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Digoxigenin with ab320717 at 1/1000 (0.503 ug/ml) dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Positive staining on (A) human tonsil incubated with mouse monoclonal PD-1 antibody conjugated to Digoxigenin and no staining on (B) human tonsil incubated with mouse monoclonal PD-1 antibody and (C) human tonsil incubated with PBS then all the sections were incubated with ab320717 at 4°C overnight.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer pH 9.0)

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Digoxigenin antibody [EPR26829-63] (AB320717)

Immunohistochemical analysis of 4% PFA-fixed 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh frozen) tissue labeling Digoxigenin with ab320717 at 1/500 (1.006 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining on (A) mouse spleen incubated with Rat Recombinant Monoclonal CD8 alpha antibody conjugated to Digoxigenin and no staining on (B) mouse spleen incubated with Rat Recombinant Monoclonal CD8 alpha antibody (ab308265) and (C) mouse spleen incubated with PBS. The nuclear counterstain was DAPI (Blue). The section was incubated with ab320717 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• cELISA

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Competitive ELISA - Anti-Digoxigenin antibody [EPR26829-63] (AB320717)

0.1 μg/mL Digoxigenin-crosslink-BSA was coated onto a 96-well plate followed by addition of a serial dilution of free Digoxigenin Testosterone or Estradiol as indicated (0-64 ng/mL). 0.1 μg/mL ab320717 (50 μL) was then added into each well before the plate was incubated for 30 minutes and washed. 1 : 20000 diluted GAR Fc-HRP was added and incubated for 30 minutes before washing again. TMB (100 μL) was added and incubated for 5-10 minutes without shaking for color development. Finally 100 μL of stop solution was added and the OD read at 450 nm on a microplate reader within 5-10 minutes.

• WB

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Western blot - Anti-Digoxigenin antibody [EPR26829-63] (AB320717)

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

This Western blot demonstrates that ab320717 specifically recognizes Digoxigenin in a competitive manner.

Exposure time : Lane 1-3 : 15 seconds; Lane 4-5 : 180 seconds

All lanes:

Western blot - Anti-Digoxigenin antibody [EPR26829-63] (ab320717) at 1/1000 dilution

Lane 1:

Digoxigenin cross-linked to BSA , blocked with 5% milk at 20 µg

Lane 2:

Digoxigenin cross-linked to BSA , blocked with Testosterone at 20 µg

Lane 3:

Digoxigenin cross-linked to BSA , blocked with Estradiol at 20 µg

Lane 4:

Digoxigenin cross-linked to BSA , blocked with Digoxigenin at 20 µg

Lane 5:

BSA , blocked with 5% milk at 20 µg

Secondary

All lanes:

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

Observed band size: 55 kDa

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