IHC image of Tau Alzheimer’s Disease staining in a section of formalin-fixed paraffin-embedded human Alzheimer brain performed on a Leica BOND^TM 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 20mins. The section was then incubated with ab246808, 1ug/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. The inset secondary-only 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.

GT-38 detects Alzheimer's disease (AD) tau pathology. Immunofluorescence staining with GT-38 (green) at 2 µg/mL followed by anti mouse-488 conjugated secondary detection antibody in 6 µm thick sections of paraffin embedded human AD hippocampus fixed in ethanol. Blue stain is DAPI positive nuclei.

GT-38 ELISA selectively detects AD-tau compared to tau monomer. GT-38 immobilized antibody selectively captures pathological tau derived from human AD brain tissue compared to recombinant tau monomer. GT-38 captured AD-tau antigens are detected with total tau rabbit polyclonal antibodies followed by HRP-conjugated anti-rabbit secondary and colorimetric readout. Total tau ELISA serves as a loading control demonstrating equivalent loading of total tau protein from both AD-tau and tau monomer.

Purified GT-38 mouse IgG1 antibody was coated on a 384 well plate at 450 ng/well (30 uL of 15 ng/uL) in 0.1 M NaHCO3 pH 9.6 at 4°C overnight. Plates were washed with PBST (phosphate buffered saline pH 7.2 with 0.05% Tween20) five times. GT-38 capture antibody was blocked with a commercial blocking solution 4°C overnight. Antigens consisted of either human brain derived insoluble tau enriched extract (AD-tau) or purified recombinant tau monomer. AD-tau was sonicated with a probe sonicator and antigens diluted in 0.2% BSA in PBS and applied to immobilized GT-38 and allowed to incubate 4°C overnight. Plates were washed with PBST five times. Rabbit polyclonal total tau detection antibody was diluted to 3.7 μg/mL in Buffer C (20 mM sodium phosphate pH 7.0, 2 mM EDTA, 400 mM sodium chloride, 1% BSA, 0.005% thimerosal), 30 μL was added to each well and incubated for 1 hour at room temperature. Plates were washed with PBST five times. Anti-rabbit HRP conjugated was diluted 1: 3,000 in Buffer C, 30 μL was added to each well and incubated for 1 hour at RT. Plates were washed with PBST five times. Colorimetric HRP substrate (30 μL/well) was added to the plate and developed at RT for 10 minutes. The reaction was quenched with addition of 30 μL/well 10% H₂PO₄ and the absorbance was measured at wavelength 450 nm.

IHC image of Tau Alzheimer’s Disease staining in a section of formalin-fixed paraffin-embedded human normal brain performed on a Leica BOND^TM 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 20mins. The section was then incubated with ab246808, 1ug/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. The inset secondary-only 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.

IHC image of Tau Alzheimer’s Disease staining in a section of frozen human Alzheimer brain performed on a Leica BOND^TM system using the standard protocol F. The section was incubated with ab246808, 1ug/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. The inset secondary-only 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.