Huab25 hrpo

For detection of human and mouse Aβ, an in-house ELISA sandwich was carried out. Briefly, 96-wells Nunc-Immuno plates (Nunc, Denmark) were coated overnight at 4 °C with JRF AB038 antibody for Aβ1‐38, JRF cAb040/28 antibody for Aβ40 or JRF Ab042/26 antibody for Aß42 (Janssen Pharmaceutica), all used at 1.5 mg/ml in PBS containing 0.1% casein (Casein Buffer). Plates were washed 5 times with Washing Buffer (PBS-0.05% Tween 20) before the addition of the samples or the standard curve made with consecutive dilutions (from 100 to 0.0003 ng/ml) of human or mouse Aβ40 and Aβ42 (rPeptide). Detection antibody was obtained from Janssen; huAB25‐HRPO. After overnight incubation at 4 °C and 5 time washes with the Washing Buffer, the samples were developed with a 0.02% TMB (tetramethylbenzidine) solution in Sodium Acetate (100 mM pH 4.9) containing 0.03% H₂O₂. The reaction was stopped with 0.2 N H₂SO₄ and read at 450 nm on a Perkin Elmer Envision 2103 multilabel reader.

Rabbit polyclonal antibodies against Psen1‐NTF (B19), Aph1a (B80.3), Nct (9C3), Pen‐2 (B126), and App C‐terminus (B63.3) have been described (Annaert et al, 2001; Esselens et al, 2004). Aph1bc (L82) was generated in the laboratory by immunizing rabbits with a QDKNFLLYNQRSR peptide. Aplp1 (W1CT) and Aplpl2 (W2CT) were a gift from D. Walsh, and Syndecan3 (2E9) was a gift from G. David. Commercially available antibodies were as follows: anti‐Psen2‐CTF (D30G3) from Cell Signaling, anti‐beta‐actin (A5441) from Sigma, Lrp1 (EPR3724) from Epitomics, Dcc (A20) and Nrg1 (F‐20) from Santa Cruz, and N‐cadherin (32/N‐cadherin) from BD. For immunohistochemistry, the same App‐CTFs were used (1:7,500), and further, NeuN (1:1,000, Chemicon Millipore clone A60 #MAB377, mouse monoclonal), Iba1 (1:900, Wako #019‐19741, rabbit polyclonal), and Gfap (1:1,000, Dako #Z0334, rabbit polyclonal). ELISA‐capturing antibodies were as follows: JRF/cAb40/28 for Aβ₄₀ and JRF/cAb42/26 for Aβ₄₂ from Janssen Pharmaceutica (Beerse, Belgium). Detection antibody huAB25‐HRPO was obtained from Janssen Pharmaceutica.