Expression of Antimicrobial Peptides: Materials and methods part 2

17 Jan
2011

Electrophoresis

The products were run on 1.5% agarose gel contain 1 yg ethidium bromide per millimeter. 20 yl of reaction mixture was mixed with loading buffer, separated by electrophoresis for 15 minutes at 100 voltages and visualized by UV transillumination.

Quantitative analysis

PCR products of hBD-2, LL-37 were normalized with GAPDH on DIG chemiluminescent film by using densitometer (volume of hBD-2/volume of GAPDH x 100, volume of LL-37/volume of GAPDH

Western blotting

The tissue were cut (hash) by scissor. Skin were lysed in a buffer containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 yg/ml phe- nylmethanesulfonyl fluoride (PMSF), 1 yg/ml apro- tinin, 1% Triton x 100, the tissue was homogenized by homogenizer. After centrifuged with 12,000 rpm at 4° for 30 minutes. The supernatant was tran­sferred into new tube, 30 yg of soluble protein were loaded in 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with sample buffer containing 1 M Tris, glycerol 50%, samples were heated at 95° for 5 minutes prior to gel loading. For hBD-2 detection, separated protein on gel electrophoresis was transferred to nitrocellulose membrane (Osmonics, Minnesota, MN, USA) at 0.16 A for 1 hour. The membrane were washed 3 times with Tris-buffered saline tween 20 (TBST), and blocked with 5% skim milk for 1 hour at room temperature. Following this, the membrane were incubated overnight at 4° with goat anti human hBD-2 polyclonal antibody (1:1500 in 5% bovine serum albumin, SantaCruz, Delaware, CA, USA) and goat anti human LL-37 polyclonal antibody (1:1500 in 5% bovine serum albumin, SantaCruz, Delaware, CA, USA) and then washed 3 times with TBST. The secondary mouse anti-goat peroxidase conjugated antibody (1:2000 in blocking solution, SantaCruz, Delaware, CA, USA) was incubated for 1 hour at room temperature. After washing the membrane with TBST, the membrane was developed with ECL solution (SantaCruz, Delaware, CA, USA) for 3 minutes then exposed to X-ray film (Roche, Indianapolis, IN, USA). Apcalis Oral Jelly

Immunohistochemistry (IHC)

IHC were carried out using sections of frozen tissues of uninvolved and involved sites in AD. In brief, 4 ym thick sections were deparaffinized in xylene three times for 5 minutes each, and epitopes were retrieved by autoclaving (121° ) for 10 minutes in citrate-buffered saline (pH 6.0). After 20 minutes of cooling at room temperature, the activity of endogenous peroxidase was quenched by treat­ment with 3% H2O2 for 5 minutes. The sections were blocked with normal goat serum for 1 hour, and incubated with mouse anti-human LL-37 and hBD-2 polyclonal antibody, respectively. After five washes with PBS, the sections were incubated with peroxidase- conjugated anti-mouse secondary antibody, FITC- anti-mouse secondary antibody and color was devel­oped with diaminobenzidine.

Statistical analysis

The amounts of hBD-2 and LL-37 for skin between involved skin and noninvolved skin were statistically compared by t-test.  cialis 10 mg

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