IHC image of beta Amyloid staining in Human Brain Alzheimer formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab205340, 0.1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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

Dot blot analysis of beta Amyloid labeled with ab205340 at 1/6000 dilution.

Lane 1: beta Amyloid (Aβ) 1-40;

Lane 2: beta Amyloid (Aβ) 1-42.

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/30000 was used as secondary antibody.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 30 seconds.

Note: Antibody reactivity was assessed using a dot blot, which is a non-quantitative method that maintains the native conformation of beta Amyloid.  beta Amyloid 1-40 and 1-42 peptides underwent the following aggregation conditions before being spotted onto a nitrocellulose membrane and detected using ab205340:

Monomers: 0.3 mg of beta Amyloid peptide was dissolved in 30 µl 100 mM NaOH and incubated at room temperature for 10 minutes. It was then diluted with 970 µl of 1% SDS and boiled for five minutes.

Oligomers: 0.3 mg of beta Amyloid peptide was dissolved in 30 µl 100 mM NaOH and incubated at room temperature for 10 minutes. It was then diluted with 970 µl of 10 mM phosphate buffer pH 7.4 containing 0.02% sodium azide and incubated at room temperature for four days.

Fibrils: 0.3 mg of beta Amyloid peptide was dissolved in 1 ml 50% hexafluoroisopropanol (HFIP) with 0.02% sodium azide. It was then stirred constantly for nine days; the first seven with a cap on and the final two with the cap removed to allow evaporation of the HFIP. Fibrils were then sedimented at 20,000 rpm in a microcentrifuge for 20 minutes and resuspended in 1 ml of PBS + 0.02% sodium azide.

Negative control (secondary ab only) Dot blot analysis of beta Amyloid.

Lane 1: beta Amyloid (Aβ) 1-40;

Lane 2: beta Amyloid (Aβ) 1-42.

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/30000 was used as secondary antibody.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 30 seconds.