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 beta tubulin (ab6046, 0.5µg/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, 0.125µg/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.

The negative control (secondary antibody only, no primary) inset shows no staining, demonstrating secondary antibody specificity.

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

All lanes : Anti-NRG1 type III antibody (ab23248) at 1 µg/ml

Lanes 1 & 3 : Mouse Brain Tissue Lysate
Lanes 2 & 4 : Rat Brain Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 0.05 µg/ml
Lanes 3-4 : Anti-Rabbit IgG VHH Single Domain (HRP) (ab191866) at 0.05 µg/ml

Developed using the ECL technique.

Performed under reducing conditions.

IHC image of Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human cerebellum 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 monoclonal antibody [EPR12763] to NeuN (ab177487, 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, 1.0µg/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.

The negative control (secondary antibody only, no primary) inset shows no staining, demonstrating secondary antibody specificity.

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

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 was ab97051, 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.

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, 0.025µg/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.

The negative control (secondary antibody only, no primary) inset shows no staining, demonstrating secondary antibody specificity.

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

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 VDAC1 (ab15895, 1/1000 dilution) 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, 0.125µg/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.

The negative control (secondary antibody only, no primary) insert shows no staining, demonstrating secondary antibody specificity.

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

All lanes : Anti-GAPDH antibody - Loading Control (ab37168) at 1 µg/ml

Lanes 1 & 3 : HeLa Whole Cell Lysate
Lanes 2 & 4 : Jurkat Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 0.02 µg/ml
Lanes 3-4 : Anti-Rabbit IgG VHH Single Domain (HRP) (ab191866) at 0.02 µg/ml

Developed using the ECL technique.

Performed under reducing conditions.