Anti inos antibody

Equal amounts of protein/cell lysate obtained from mycobacterial-stimulated cultures or slices (10 μm) of frozen patient skin biopsies (BT, n = 3 and LL, n = 3) were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Immunoblottings were developed with rabbit polyclonal anti-iNOS antibody (Santa Cruz), rabbit polyclonal anti-SOCS3 (Santa Cruz), rabbit polyclonal anti-pSTAT1 (Cell signaling Technology, Danvers, MA, USA), mouse monoclonal anti-STAT1 (Cell Signaling Technology), and anti-β-tubulin (Santa Cruz) in 5% (w/v) nonfat milk dissolved in Tris-Tween-buffered saline (TTBS; 20 mMTris-HCl buffer, pH 7.6, containing 137 mM NaCl–0.05% [v/v] Tween 20). Results were visualized via an enhanced chemiluminescence detection system (ECL; Amersham Biosciences, Psicataway, NJ, USA).

Proteins were extracted from THP-1 transfected with miR-155 mimic or miR-155 inhibitor; HVSMC treated with supernatant of the transfection cells as above and tumor tissue from mouse. Western blotting was performed to assess the rabbit anti-MMP2 antibody (1:1000; Thermo Scientific, U.S.A.), goat anti-MMP9 antibody (1:500; Santa Cruz Biotechnology, U.S.A.), rabbit anti-monocyte chemoattractant protein (MCP-1) antibody (1:500; Bio-Rad Laboratories, U.S.A.), anti-iNOS antibody (1:100, Santa Cruz), anti-TNFα (1:1000, Sigma-Aldrich), anti-α-SMA antibody (1:1000; Millipore U.S.A.), and anti-Osteopontin antibody (1:500; Millipore, U.S.A.). Briefly, proteins (30 µg) were separated using 12% SDS gel electrophoresis and electrotransferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, U.S.A.). Then membranes were blocked with 5% nonfat dry milk in TBS-Tween for 1 h at room temperature, and incubated with primary antibodies at 4°C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibody was used at a 1:5000 dilution (DAKO) for 2 h at room temperature. Bands were visualized using enhanced chemiluminescence (ECL Advance™; GE Healthcare). Protein expression was quantified with densitometric analysis using the ChemiDoc™ imaging system (Bio-Rad Laboratories) and QuantityOne™1-D Analysis Software (Bio-Rad Laboratories).

Fingolimod (FTY720) HCl was purchased from Selleckchem (Houston, TX, USA) and anti-Iba1 antibody was purchased from FUJI FILM Wako Pure Chemicals (Japan). Anti-iNOS antibody was purchased from Santa Cruz Biotechnology, while anti-Mannose Receptor (CD206) antibodies were purchased from Abcam. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), Phospho-MEK1/2 (Ser217/221), MEK1/2, Phospho-JNK (Thr183/Tyr185), JNK, Phospho-p38MAPK (Thr180/Tyr182), p38 MAPK, and Na/K-ATPase antibodies were purchased from Cell Signaling Technology. The S1PR1 and S1PR3 antagonist VPC 23019 was purchased from Avanti Polar Lipids (Alabaster, USA).

Immunostaining was performed on serial thin sections of paraffin blocks (4 μm thickness). Xylene was used to deparaffinize tissue sections. They were rehydrated in ethanol then treated with hydrogen peroxide 3% for 10 min to exclude nonspecific reaction. Microwave antigen retrieval was performed in citrate buffer 0.01 M (pH 6.0) for 15–20 min. The slides washed in phosphate buffer saline (PBS) were incubated with anti-iNOS antibody (dilution 1: 50; Santa Cruz Biotechnology, USA) for 60 min at room temperature. Binding site of primary antibodies was visualized by Dako En Vision ™ kit (Dako, Copenhagen, Denmark). The tissue sections were incubated in diaminobenzidine (DAB) for 15 min then counterstained with Mayer’s hematoxylin to visualize the immunohistochemical reaction. The evaluation of iNOS staining was done by assessment the proportion of cells expressing iNOS as a continuous percentage (0%–100%) scale using light microscopy at high magnification (× 40). A score of +3 was considered positive (25 (link)).

For immunohistochemical, hippocampus sections were incubated overnight with anti-iNOS antibody (Santa Cruz Biotechnology, 1 : 500 in PBS, v/v). Sections were then washed with PBS and incubated with secondary antibodies. For quantitative analysis, original immunohistochemical photographs were assessed by densitometer using MacBiophotonics ImageJ 1.41a software [38 (link), 39 (link)].

The immunofluorescence analysis was carried out according to previously published methodology [50 (link)]. Anti-68 antibody (Santa Cruz Biotechnology) and anti-iNOS antibody (Santa Cruz Biotechnology) and anti-Arg-1 antibody (Santa Cruz Biotechnology) were incubated in a humidified chamber at 37 °C O/N on the sections. After washing with PBS, sections were incubated for 1 h at 37 °C with secondary antibodies, TEXAS RED-conjugated anti-rabbit Alexa Fluor-594 (Molecular Probes, Eugene, OR, USA) and FITC-conjugated anti-mouse Alexa Fluor-488 (Molecular Probes, Eugene, OR, USA). Nuclei were stained by adding 2 μg/mL 40,60-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS. Slides were then washed with PBS and incubated with a secondary antibody. Specific labeling was identified with an avidin–biotin-peroxidase complex and a biotin-conjugated goat anti-rabbit immunoglobulin G (Vector Lab, Milan, Italy) [51 (link)]. Stained sections were observed using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy). Each specimen was observed in five random visual fields and the average number of double-positive cells in each specimen was calculated [19 (link)].