Liquid dab chromogen

An EnVision⁺ kit/HRP kit (Dako, Carpinteria, CA, USA) was used. Paraffin‐embedded sections of lung tumor specimens were processed in a microwave (90 °C) with antigen retrieval solution (pH 9) (S2367; Dako), treated with a peroxidase‐blocking reagent, and then treated with a protein‐blocking reagent (K130, X0909; Dako). Tissue sections were incubated first with a rabbit anti‐EHMT1 antibody (BIO MATRIX RESEARCH, Chiba, Japan) and then with an HRP‐conjugated secondary antibody (Dako). Immunoreactivity was visualized with a chromogenic substrate (Liquid DAB Chromogen; Dako). Finally, tissue specimens were stained with Mayer’s hematoxylin solution (Hematoxylin QS; Vector Laboratories, Burlingame, CA, USA) for 20 s to distinguish nuclei from the cytoplasm. Human lung cancer tissues were purchased from BioChain [11 (link)].

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of Acacia pollen extract (60 μg) was performed according to Laemmli [15 (link)] using 12.5% or 15% acrylamide separation gels under reducing and nonreducing conditions. Reducing and nonreducing sample buffers were the same except that the final reducing sample buffer contained 5% (vol/vol) 2-mercaptoethanol (2-ME). Separated protein bands from the electrophoresis of A. farnesiana pollen extract were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore Corp., Bedford, MA, US), as described earlier [10 (link)]. In brief, after washing and blocking, membranes were incubated with a 1/5 dilution of serum pool or individual sera from patients with A. farnesiana allergy or with control sera (1 : 5 dilutions). Biotinylated anti-human IgE (Nordic Immunology Co., Netherlands) (1 : 500 v/v in 1% BSA) was added to the blotted membrane strips and incubated for 2 h at room temperature. The unbound antibodies were removed from blots by washing with PBS and followed by incubation with 1 : 10000 v/v in BSA1% HRP-linked streptavidin (Sigma-Aldrich, USA) for 2 h at room temperature. The bound enzymatic activity of horseradish peroxidase was detected by high-sensitivity liquid diaminobenzidine (Liquid DAB⁺) chromogen (DAKO, Denmark).