Anti t bet

The right upper lung tissues were homogenized in the presence of protease inhibitors, and the protein concentrations were determined using NE-PER® nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, USA). The total proteins (50 μg) were loaded onto SDS-PAGE gels. After electrophoresis at 120 V for 90 min, the separated proteins were transferred to polyvinylidene difluoride membranes using a wet transfer method (250 mA for 90 min) [21 (link)]. Nonspecific sites were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, and the membranes were then incubated overnight at 4°C with anti-GATA-3, anti-T-bet (Santa Cruz Biotechnology Inc., USA), or anti-HDAC1–11 (HDAC Family Antibody Set; BioVision, USA) primary antibodies. Horseradish peroxidase-conjugated anti-rabbit IgG was used to detect binding of the primary antibodies. The membranes were stripped and reprobed with an anti-actin primary antibody (Sigma-Aldrich, USA) to verify equal protein loading in each lane. The binding of the specific antibodies was visualized by exposure to photographic film. The densities of the stained bands for T-bet, GATA-3, and HDAC1–11 relative to the staining band for β-actin were quantified with Quantity ONE densitometry software (PDI Imageware Systems, USA).

Slices with 4 µm thickness were arranged on silanized slides (3-aminopropyltriethoxysilane; Sigma-Aldrich Inc., St. Louis, MO, USA), deparaffinized in a 60°C chamber, sequentially hydrated in passages through xylol, absolute alcohol, 70% alcohol, and distilled water. The avidin-biotin peroxidase-anti-peroxidase complex method was used for sample staining. Tissue samples were immersed in 1 mM citrate buffer, PH 6.0, blocked with 3% hydrogen peroxide, and incubated with primary antibodies (polyclonal rabbit anti-iNOS, anti-Nrf2, anti-T-bet, and anti-IL17, diluted 1:100—Santa Cruz, CA) for 1 h. Next, samples were incubated with biotinylated secondary antibodies for 30 min and the avidin-biotin complex for an additional 30 min. Samples were stained by addition of the diaminobenzidine chromogen (DAB) substrate for ∼1 min. The negative control were carried out by omitting incubation with primary antibodies. Tissue sections were examined by light microscopy (400×), and 10 photomicrographs were collected per section (Zeiss Axiostar, Zeiss, Germany).