Donkey Anti-Rabbit IgG H&L (Biotin) (ab207999)

IHC image of Histone H4 staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. Ab207999 Donkey Anti-Rabbit IgG H & L (Biotin) was used as the secondary antibody.

Staining was performed on a Leica Bond^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins, before blocking of endogenous biotin using ab64212. The section was then incubated with ab177840, 1/100 dilution, for 15 mins at room temperature, followed by ab207999, 1/2000 dilution, for 15 mins at room temperature. Detection was via an HRP conjugated ABC system and DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab207999 was tested by direct ELISA, where wells were coated with serially diluted rabbit IgG (1000 – 16 ng/ml) for 2 hours, followed by a 2 hour blocking step (5% BSA). ab207999 (1:20,000 dilution; 2 hours) was added and detected by streptavidin-HRP (ab7403; 1:10,000 dilution; 1 hour). Signal was developed by TMB substrate. Data from duplicates; +/- SD.

IHC image of beta Tubulin staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. Ab207999 Donkey Anti-Rabbit IgG H & L (Biotin) was used as the secondary antibody.

Staining was performed on a Leica Bond^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins, before blocking of endogenous biotin using ab64212. The section was then incubated with ab6046, 1/100 dilution, for 15 mins at room temperature, followed by ab207999, 1/2000 dilution, for 15 mins at room temperature. Detection was via an HRP conjugated ABC system and DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Cross-reactivity of the polyclonal secondary antibody ab182020 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 µg/ml (50 µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. ab182020 was then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Goat anti-Donkey IgG H&L (HRP) (ab6988) was used at 1/20,000 dilution (50 µl/well), followed by incubation for 1h at RT.

For the batch tested, ab182020 showed a cross-reactivity below 2% towards human IgG, mouse IgG, rat IgG, goat IgG and chicken IgY.

This data was developed using the unconjugated antibody (ab182020).