Donkey Anti-Goat IgG H&L (Biotin) (ab208000)

IHC image of alpha Tubulin staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. Ab208000 Donkey Anti-Goat 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 ab7291, 1/100 dilution, for 15 mins at room temperature. Ab98800 (Goat anti-Mouse IgG) was added as a bridging antibody at 1/250 dilution for 1 hour, followed by ab208000, 1/500 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

ab208000 was tested by direct ELISA, where wells were coated with serially diluted goat IgG (1000 – 16 ng/ml) for 2 hours, followed by a 2 hour blocking step (5% BSA). ab208000 (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.