Anti hbd 2 antibody

Formalin-fixed, paraffin-embedded skin tissues (3 samples of tuberculosis, 2 samples of tuberculids, and 10 samples of normal skins) were used for the immunohistochemical studies. For immunoperoxidase staining, sectioned tissues were treated for endogenous peroxidase inactivation (3% hydrogen peroxide). Then, the tissues were blocked. Specimens were then incubated overnight with primary antibody. Anti-HBD-2 antibody (Abcam, UK) was used at 1:100 dilution, anti-HBD-3 antibody (Novus Biological, USA) at 1:50 dilution, and anti-LL-37 antibody (Abcam, UK) was diluted to 1:500. Then, secondary antibody (anti-mouse/anti-rabbit antibody) was added sequentially for 30 min, followed by 3-amino-9-ethylcarbazole/hematoxylin color spectrum analysis. The result was observed by the use of DC 300F microscopic image analysis system (Leica Microsystems GmbH, Wetzlar, Germany), and the mean optical density values of three distinct groups were measured.

Skin tissues were homogenized in radioimmunoprecipitation assay buffer with phenylmethanesulfonyl fluoride on ice for 30 min, vibrated, and centrifuged. Total protein of the skin tissue lysates was evaluated by BCA protein assay (Fish Scientific, Fair Lawn, NJ, USA). Skin lysates containing 30 μg of crude skin tissue lysate protein were analyzed by NuPAGE 4–12% Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and was transferred to polyvinylidene fluoride membranes (Invitrogen). The membranes were incubated overnight with the polyclonal rabbit anti-HBD-2 antibody (Abcam), anti-HBD-3 antibody (Novus Biological, USA), or anti-LL-37 antibody (Abcam) at 1:1000 dilution, respectively. The analysis was performed with chemiluminescence reagents. Protein expression was normalized to the quantity of beta-actin. The signal and grayscale values were visualized and analyzed by using ImageJ software (GE Healthcare Piscataway, NJ, USA), and grayscale value ratios were calculated.