Streptavidin peroxidase polymer ultrasensitive

Peptides in 5 mg/ml stock solution in 0.01 M AcOH were diluted to 5 μg/ml in PBS (pH 7.4) unless stated otherwise (for concentration studies, peptides were diluted to between 0.1 and 10 μg/ml as indicated). 100 μl of peptide solution was added to wells containing collagen films and incubated for 30 min in the dark at room temperature. Preliminary experiments indicated that incubation of peptide with a collagen scaffold for 30 min was optimal for passive absorption of a similar peptide (L. Mullen, PhD Thesis, University of Cambridge, 2010), and this incubation time was adopted for the experiments described here. Wells were then placed under a long-wavelength UV lamp (Blak-Ray B100AP, 365 nm wavelength) for 2–60 min, and routinely for 5 min, as indicated. Negative control wells were covered in aluminium foil to restrict UV exposure. Following UV treatment, wells were washed 3 times 2 min with 200 μl citrate buffer (pH 3) containing 1 mg/ml BSA and three times with 200 μl of PBS containing 1 mg/ml BSA. 100 μl of Streptavidin-Peroxidase Polymer Ultrasensitive (Sigma #S2438) diluted in PBS to 1:10000 were added to the wells and incubated 45 min at room temperature. After washing four times with 200 μl of PBS containing 1 mg/ml BSA, 100 μl of TMB substrate was added, the reaction stopped with 100 μl of 2.5 M sulphuric acid and A₄₅₀ was measured.

IHC studies were performed on 20 patients with DRE for the analysis of VEGF-A content in brain capillary endotheliocytes. Biopsies of the temporal lobe were fixed with 10% paraformaldehyde in 0.1 M sodium phosphate buffer, dehydrated in a standard manner, and embedded in paraffin. IHC reactions were performed on paraffin-embedded 5–7 μm thick slices of the brain temporal lobe biopsies according to the standard protocol. The VEGF-A antibody (Anti-VEGF Receptor 2 antibody [SP123] Abcam, Boston, MA, USA) was used as a primary antibody. For visualization, the Streptavidin-Peroxidase Polymer Ultrasensitive system (Streptavidin-Peroxidase Polymer, Ultrasensitive, Product Number: S 2438, Sigma-Aldrich, St. Louis, MO, USA) and DAB chromogen (3,3′-Diaminobenzidine tetrahydrochloride hydrate, Product Number: D5637, Sigma-Aldrich, USA) were used. The sections were counterstained with Gill’s hematoxylin, and embedded in Bio Mount HM synthetic embedding medium (Bio Mount HM Mounting medium, Catalog number: 05-BMHM100, BIO-OPTICA Milano, Italy). Additionally, reactions lacking primary antibodies were performed to ensure the specificity of the observed staining. Sections were analyzed with a light microscope (Zeiss Mirax Midi BF/FL Fluorescence Slide Scanner Imaging System, Carl Zeiss MicroImaging GmbH., Jena, Germany).

Recombinant human TrkA-Fc chimera (125 ng/50 μl; R&D Systems, 175-TK) was coated onto NUNC F96 Maxisorp ELISA plates (Sigma-Aldrich) at 4 °C overnight. The plate was washed 3x in PBST and 2x PBS and then blocked for 1 hr at room temperature with Blocker^TM 1% casein in PBS (Thermo Scientific #37528), followed by 4x washes with PBST and 2x PBS. Thirty ng recombinant human β-NGF (R&D Systems, 256-GF/CF) was mixed with 50 µl peptide (Isogenica or Alta Biosciences, UK) at appropriate concentration in 0.1% casein in PBS/0.1% Tween. NGF/peptide solutions (55 µl/well) were transferred into the NUNC Maxisorp plate and incubated for 40 minutes at room temperature with 50 µl/well 1:200 diluted biotinylated anti-human β-NGF antibody (R&D Systems, 256-GF/CF) in 0.1% casein in PBS/0.1% Tween. The plate was washed 4x in PBST and 2x PBS. Streptavidin-HRP (Sigma-Aldrich; Streptavidin-Peroxidase Polymer, Ultrasensitive, #2438; 1:1000 dilution, 50 µl/well) in 0.1% casein/PBST was incubated on the plate for 30 minutes at room temperature followed by 4x washes with PBST and 2x PBS. TMB reagent (Thermo Scientific #34028; 50 µl/well) was incubated on the plate for 5–20 minutes, quenched with 50 µl/well 0.5 M H₂SO₄ and the absorbance read at 450 nm.