Biorad chemidoc xrs software

Extracts of the cytosol and nucleus were prepared, as previously mentioned [82 (link),83 (link),84 (link),85 (link),86 (link)]. The following primary antibodies were used: anti-iNOS (1:500, Santa Cruz Biotechnology, #sc-7271, Dallas, TX, USA), anti-Cox-2 (1:500, Santa Cruz Biotechnology, #sc-19999), anti-FAAH (1:500, Sigma-Aldrich Corp., Milan, Italy), anti-Iκbα (1:500, Santa Cruz Biotechnology, #sc-1643), and anti-nfκb (1:500, Santa Cruz Biotechnology, #sc8414) in 1 × PBS, 5% w/v non-fat dried milk, and 0.1% Tween 20, at 4 °C overnight [87 (link),88 (link),89 (link),90 (link)]. For the cytosolic fraction, Western blots were also explored with antibody against β-actin protein (1:500, Santa Cruz Biotechnology, Dallas, TX, USA). The same methods were used for nuclear fraction with lamin A/C (1:500, Sigma-Aldrich Corp., Milan, Italy) [91 (link),92 (link)]. Signals were examined with an enhanced chemiluminescence (ECL) detection system reagent, according to the manufacturer’s instructions (Thermo, Monza, Italy). The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDoc^TM XRS+ software (Hercules, CA, USA) [83 (link),87 (link),93 (link),94 (link),95 (link)].

Cytosolic and nuclear extracts were prepared as previously described [41 (link)]. The following primary antibodies were used: anti-NF-kB (SCB; 1:500 #sc8008), anti-NRF-2 (sc-365949, 1:1000, SCB), anti-HO-1 (sc-136960, 1:1000 SCB), anti-Bcl-2 (SCB, sc-7382), anti-Bax (SCB, sc-7480), in phosphate-buffered saline, 5% w/v non-fat dried milk, and 0.1% Tween-20 at 4 °C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA; 1:2000) for 1 h at room temperature. Anti-β-actin or anti-lamin A/C antibodies were used as controls. The expression of protein bands was detected by a procedure previously described [41 (link)]. To establish that the blots were loaded with identical volumes of lysate, they were also probed with anti-β-actin or anti-lamin A/C antibodies. Comparative expression of the protein bands was identified with a chemiluminescence detection procedure, following the manufacturer’s instructions (Super Signal West Pico Chemiluminescent Substrate; Pierce). The expression of protein bands was computed by densitometry with BIORAD ChemiDocTM XRS+software and standardized to β-actin or lamin A/C levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003).

Western blot analysis was performed as previously described [36 (link),37 (link),38 (link)]. Briefly, zebrafish larvae were homogenized in ice-cold RIPA buffer to extract proteins. Each set of larvae (20 per experimental group of each experiment) was pooled for protein preparation such that n = 1 refers to protein from these 20 larvae. Protein concentrations were determined by the BCA method [39 (link),40 (link)]. After electrophoresis, the proteins were transferred from gels onto a polyvinylidene fluoride (PVDF) membrane 0.42 µm (GE Amersham, Casoria NA). Primary antibodies were incubated at 4 °C overnight, and antibody against Bax (Abcam ab32503, 1:800), bcl-2 (Abcam ab18285, 1:800), iNOS (Antibodies A323357, 1:500), and the proteins expression were normalized according to the expression of GAPDH (Abcam ab181602, 1:1000). After being washed three times with TBST, the membrane was incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit lgG or goat anti-mouse lgG (diluted at 1:5000) for 2 h at room temperature. Finally, the immunoreactive bands were detected using the ECL methods, and the protein bands were quantified by densitometry with BIORAD ChemiDocTM XRS+ software (Bio Rad, Hercules, CA, USA). The protein expressions were obtained by analyzing the density ratio of target proteins to GAPDH expression.

Western blot examination on cytosolic fraction of the paw tissue was prepared as previously described [59 (link)]. Membranes were incubated with anti-5-LOX (1:1000) (Santa Cruz Biotechnology, Heidelberg, Germany), anti-Cox-2 (1:1000) (Santa Cruz Biotechnology, Heidelberg, Germany), and β-actin (1:500) (Santa Cruz Biotechnology, Heidelberg, Germany) for the standardization. Signals were identified with enhanced chemiluminescence (ECL) detection system reagent and the relative expression of the protein bands was measured by densitometry with BIORAD ChemiDocTM XRS+software (Bio-rad, Milan, Italy). A representation of blot signals were imported to analysis software (Image Quant TL, v2003).

Extracts of the cytosol and nucleus were prepared, as previously mentioned [84 (link),87 (link),88 (link),89 (link),90 (link)]. The following primary antibodies were used: anti-Bax (1:500, Santa Cruz Biotechnology, #sc7480), anti-Bcl-2 (1:500, Santa Cruz Biotechnology, #sc7382), anti-Iκbα (1:500, Santa Cruz Biotechnology, #sc-1643), and anti-nfκb (1:500, Santa Cruz Biotechnology, #sc8414) in 1× PBS, 5% w/v non-fat dried milk, and 0.1% Tween 20, at 4 °C overnight [91 (link),92 (link),93 (link),94 (link)]. For the cytosolic fraction, Western blots were also probed with antibody against β-actin protein (1:500, Santa Cruz Biotechnology, Dallas, TX, USA). The same methods were used for nuclear fraction with lamin A/C (1:500, Sigma-Aldrich Corp., Milan, Italy) [95 (link),96 (link)]. Signals were examined with an enhanced chemiluminescence (ECL) detection system reagent, according to the manufacturer’s instructions (Thermo, Monza, Italy). The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTM XRS+ software [84 (link),91 (link),97 (link),98 (link),99 (link)].

Cytosolic and nuclear extracts were prepared as previously described [72 (link),73 (link)]. The following primary antibodies were used: anti-NRF-2 (1:500, Santa Cruz Biotechnology, Heidelberg, Germany, #sc-365949), anti-Heme Oxigenase 1 (HO-1; 1:500, Santa Cruz Biotechnology, Heidelberg, Germany, #sc-136960), anti-Bax (1:500, Santa Cruz Biotechnology, #sc7480), and anti-Bcl-2 (1:500, Santa Cruz Biotechnology, #sc7382) in 1× PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20 at 4 °C overnight. To ensure that blots were loaded with equal amounts of proteins, they were also probed with antibodies against the β-actin protein for cytosolic fraction (1:500; Santa Cruz Biotechnology, Heidelberg, Germany,) or lamin A/C for nuclear fraction (1:500 Sigma-Aldrich Corp., Milan, Italy). Signals were examined with an enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions (Thermo, Monza, Italy). The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTM XRS⁺ software and standardized to the β-actin and lamin A/C levels.