Rabbit polyclonal anti tgf β1

After fixation of gastric tissues in 10% formalin saline overnight, the tissues were embedded in paraffin bee wax at 56 °C for 6 h. Paraffin wax tissues were cut by rotary microtome at 5 µm thickness. Then the sections were deparaffinized and were washed with tap water and dehydrated in serial dilutions of ethyl alcohol. Then the specimens were cleared in xylene and placed in 0.3% hydrogen peroxide/methanol for 20 min to block endogenous hydrogen peroxidase activity followed by washing with PBS. After that, 10% normal goat serum was added followed by incubation overnight at 4 °C in a humid chamber with mouse monoclonal anti- PCNA (1:75) (Santa Cruz Biotechnology), rabbit polyclonal anti-TGF-β1 (1:500) (Abcam, USA), and anti-EGF (1:100) (Abcam, USA). After that, they were incubated with the biotinylated goat anti-rabbit secondary antibody (1:200, Vector Labs, Peterborough, UK) for 30 min at room temperature followed by the addition of 3,3′-diaminobenzidine (DAB) chromogen. Eventually, sections were counterstained with Mayer’s hematoxylin and examined by a light microscope. For negative control; sections were routinely processed but the primary antibodies were replaced by PBS.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 0.25% trypsin-EDTA, penicillin and streptomycin (PenStrep) were purchased from Invitrogen (Carlsbad, CA, USA). 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluoresceindiacetate, acetyl ester (CM-H2DCFDA) was obtained from Molecular Probe (Invitrogen). LPS (Escherichia coli 0127:B8) was purchased from Sigma-Aldrich (St. Louis, MO). The following primary antibodies were used in this study: rabbit monoclonal anti-Nox4,rabbit polyclonal anti-TNFα, rabbit polyclonal anti-TGFβ1 (Abcam, Cambridge, MA, USA); rabbit polyclonal anti-Nox4 (Millipore, Billerica, MA, USA); rabbit polyclonal anti-NFĸB p65, rabbit polyclonal anti-PCNA, goat polyclonal anti-GAPDH, goat polyclonal anti-actin (Santa Cruz Biotechnology, CA, USA); rabbit monoclonal anti-ERK1/2, rabbit monoclonal anti-MyD88 (Cell Signaling Technology, MA, USA); mouse monoclonal Anti-ERK1/2 (pT202/pY204) (BD Biosciences, USA) and rabbit polyclonal anti-NFĸB p65(Ser536) (Bioss Antibodies, USA).

Lung tissues were fixed and embedded routinely, and immunohistochemistry was performed with rabbit polyclonal anti-TGF-β₁ (1 : 100 vol/vol, Abcam) and rabbit monoclonal anti-α-SMA (1 : 100 vol/vol, Abcam) as primary antibodies to measure the expression of TGF-β1 and α-SMA. Tissue sections were deparaffinized and rehydrated successively, and then antigen retrieval was performed by citrate buffer. Slices were incubated with primary antibody (anti-TGF-β₁, anti-α-SMA) overnight at 4°C in wet box, rinsed in PBS 3 times, and then incubated with biotinylated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies (1 : 100, Abcam). Coloring was performed with the DAB staining method, and then slices were dewaxed and sealed. We observed and photographed the slices using an Olympus microscope. Pictures were analyzed by ImagePro plus version 6.0 for Windows. Using the Hue-Saturation-Intensity mode, we chose positive region stained into brown, and calculated IOD value for each picture.