Pierce 3 3 diaminobenzidine dab substrate kit

Protein samples were separated by 15% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, USA), which were blocked with 5% BSA (diluted in Tris buffered saline with 0.1% Tween20 buffer) overnight at 4 °C. HDM allergic serum and healthy serum samples (1:5 dilutions) were incubated with the membranes for 2.5 h at 37 °C, separately. Mouse anti-human IgE (Fc)-HRP (Southernbiotech, Cat. 9160-05, 1:1000) was added for 1 h at 37 °C after washing. The bands were visualized with a Pierce™ 3,3′ diaminobenzidine (DAB) substrate Kit (Thermo Scientific, Cat. 34002).

Paraffin-embedded tissue sections measuring 4 μm were analyzed for Trichromic and indirect immunoperoxidase staining. Immunohistochemical staining was carried out using rabbit anti-CD44 polyclonal antibody (Abcam) and anti-βIII tubulin (Cell Signaling). Briefly, samples were fixed in formalin and embedded in paraffin, subjected to deparaffinization in xylene, and dehydrated in ethanol. The block of endogenous peroxidase was performed at room temperature for 30 min, with 0.1% hydrogen peroxide–methanol. The samples were washed twice with distilled water and incubated with target retrieval solution (Dako, Glostrup, Denmark) in a microwave oven. The samples were the washed twice with distilled water. Non-specific binding was performed with a specific reagent for 15 min. The sections were incubated overnight with anti-CD44 and βIII tubulin antibodies in a dilution of 1:100, at 4 °C. Protein identification was obtained using an Horseradish peroxidase (HRP) conjugated secondary antibody IgG Super Vision Kit (Italian distributor of Boster immunoleader USA, Tema Ricerca, Bologna, Italy) and the Pierce 3,3′-Diaminobenzidine (DAB) Substrate Kit (Thermo Scientific, Italian distributor Tema Ricerca) for 5 min at room temperature. Cells were counterstained using Meier’s hematoxylin. The procedures were performed according to the protocols provided by manufacturers.