Top left: IHC image of Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human colon tissue section.
The section was dewaxed and then pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with rabbit polyclonal antibody to Ki67 (ab15580, 0.1µg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. Endogenous peroxidases were quenched using 1.6% (v/v) hydrogen peroxide in TBS containing 0.025% (v/v) Triton X-100 for 30 minutes at room temperature, with agitation. The secondary antibody, Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866, 0.1µg/ml) was then applied for 1 hour at room temperature in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA before being developed for 10 minutes at room temperature using Steady DAB/Plus (ab103723). The section was then counterstained with hematoxylin and mounted with DPX.
Top right: this image shares the same experimental design parameters, except the secondary antibody wasab97051, goat anti-rabbit IgG H&L (HRP) (0.1µg/ml). This demonstrates the improved definition of staining given by VHH Single Domain Antibodies over conventional secondaries.
Bottom right: this image shares the same experimental design parameters but is a negative control (no primary antibody) for ab97051, demonstrating specificity of the goat anti-rabbit secondary antibody.
Bottom left: this image shares the same experimental design parameters but is a negative control (no primary antibody) for ab191866, demonstrating the specificity of the VHH-Single Domain Antibody.
For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.
