Anti-beta Amyloid 1-42 antibody [mOC64] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Amyloid 1-42 antibody [mOC64] - BSA and Azide free (AB271968)

Composite multiplex immunofluorescence staining of APP, PSD95 and Tau staining in a section of formalin-fixed paraffin-embedded human Alzheimer’s brain*.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with EDTA (Ph9.0) using retrieval settings of 100°C for 40 minutes. The section was then incubated at room temperature for 1 hour with ab201060 at 1µg/ml dilution (shown in green), ab317793 at 1µg/ml (shown in magenta), and ab317841 at 1µg/ml (shown in yellow). Then incubated for 1 hour with ab150118 Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed 1/1000, ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 1/1000, and ab150135 Donkey Anti-Goat IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Dako Fluorescence Mounting Medium ^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Amyloid 1-42 antibody [mOC64] - BSA and Azide free (AB271968)

This data was developed using ab201060, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human alzheimer brain labelling beta Amyloid with ab201060 at a concentration of 0.1μg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab201060 Anti-beta Amyloid 1-42 antibody [mOC64] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Dot

Supplier Data

Dot Blot - Anti-beta Amyloid 1-42 antibody [mOC64] - BSA and Azide free (AB271968)

Dot blot analysis of human beta Amyloid 1-42 labeled with ab201060 at 1/7000 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 1/5000 dilution was used as secondary antibody.
Blocking and diluting buffer : 5% NFDM/TBST.
Exposure time : 30 seconds.

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 ab201060 :
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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201060).

• Dot

Supplier Data

Dot Blot - Anti-beta Amyloid 1-42 antibody [mOC64] - BSA and Azide free (AB271968)

Negative control (secondary ab only) :

Lane 1 : Human beta Amyloid (Aβ) 1-40.
Lane 2 : Human beta Amyloid (Aβ) 1-42.
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/5000 dilution was used as secondary antibody.
Blocking and diluting buffer : 5% NFDM/TBST.
Exposure time : 30 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201060).