Peroxidase conjugated goat anti rabbit igg

Western blots were performed as already described [56 (link)]. The specific primary antibodies anti-PCNA (sc-56, Heidelberg, Germany), anti-p-ERK (sc-7383, Heidelberg, Germany), anti-p-p38 (sc-166182, Heidelberg, Germany), anti-Nox-1 (PA5-103220), and anti-Nox-4 (PA5-72816) were mixed in a 5% w/v nonfat dried milk solution and incubated at 4 °C overnight. Blots were incubated with a peroxidase-conjugated goat antirabbit IgG (Jackson Immuno Research) or a peroxidase-conjugated bovine antimouse IgG secondary antibody for 1 h at room temperature [57 (link)]. To confirm the equal amounts of protein, filters were also incubated with the antibody against β-Actin (sc-47778). Signals were detected with an enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate) [58 (link)]. The relative expression of the protein bands was quantified using densitometry with Bio-Rad ChemiDoc XRS software 2.1.1, #1708265 [59 (link)]. Images of the blot signals were imported into analysis software (Image Quant TL, Amersham Biosciences, Freiburg, Germany, v2003) [60 (link)].

The 3T3-L1 fibroblasts were differentiated into mature adipocytes, as described above, and shock-frozen by ice-cold acetone (ROTH, Karlsruhe, Germany) for immunocytochemistry. The air-dried cells were incubated in PBS for rehydration. The endogenous peroxidase activity was blocked with 3% H₂O₂ (ROTH, Karlsruhe, Germany). To avoid non-specific protein binding, the cells were incubated in 10% bovine serum albumin (BSA, ROTH, Karlsruhe, Germany), 10% fetal calf serum (FCS, Sigma-Aldrich, Steinheim, Germany) and 10% chicken serum (Sigma-Aldrich, Steinheim, Germany), followed by 3 h incubation in a moist chamber with a polyclonal anti-TLR7 antibody from rabbit (2 µg/mL in 1% BSA; Invitrogen, Carlsbad, CA, USA). The cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (1.6 µg/mL in 1% BSA; Jackson Immuno Research, West Grove, Pennsylvania, USA) for 90 min. The color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, CA, USA) at room temperature was stopped after microscopic examination. The rabbit isotype-matched IgG sera (ab37415; Abcam, Cambridge, UK) served as an isotype control. Parallel experiments without primary antibodies were carried out as negative controls.

Western blots were performed as described in our previous studies [14 (link),36 (link)]. Specific primary antibody anti-COX-2 (1:600, Santa Cruz Biotechnology) or anti-PGE2 (1:700; Bioss Antibodies) was mixed in 1× PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20, and incubated at 4 °C, overnight. After that, blots were incubated with the peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with the antibody against beta actin (1:1000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003).

Western blots were performed as described from our previous studies [60 (link)]. Specific primary antibody: anti-NRF2 (1:600, Santa Cruz Biotechnology) or anti-iNOS (1:700; Santa Cruz Biotechnology) or anti-IkB-α (1:700; Santa Cruz Biotechnology) or anti-NF-kB (1:700; Santa Cruz Biotechnology) was mixed in 1 × PBS, 5% w/v nonfat dried milk, and 0.1% Tween 20, and were incubated at 4 °C, overnight. Afterwards, blots were incubated with peroxidase-conjugated bovine antimouse IgG secondary antibody or peroxidase-conjugated goat antirabbit IgG (1:2000, Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with the antibody against laminin (1:1000; Santa Cruz Biotechnology) and GADPH (1:1000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003).

Hypothalamus were homogenized in RIPA buffer and cleared by centrifugation, according to the standard techniques [28 (link)]. Western blots of whole tissue lysates were probed with primary antibodies against Kisspeptin1 (KISS1, Solarbio, K009431P), Kisspeptin1 receptor (KISS1R, Solarbio, K003544P), GnRH(PA5-97047, Thermo Fisher), GnRH receptor (abcam, ab183079), Tyrosine hydroxylase (TH, AB152; Merck Millipore), Arginine vasopressin (AVP, AB1565, Merck Millipore), Neuronal Nuclei (NeuN; MAB377, Merck Millipore), brain-derived neurotrophic factor (BDNF; AB203573, Abcam) and β-Tubulin (A01030HRP, Abbkine). The secondary antibody used was either peroxidase- conjugated goat anti-rabbit IgG (111-035-003; Jackson), or peroxidase-conjugated goat anti-mice IgG (115-035-003; Jackson). Protein markers (20351ES76; Shanghai Yisheng, China) were added on both sides of each gel to verify bands. The PVDF membranes were detected by enhanced che- moluminescence (Beyotime, China). Bands were analyzed using Image Lab™ Software (Bio-Rab Laboratories), were normalized to β-Tubulin and expressed as relative units (RU).

Immunohistochemistry was performed as previously described (Paterniti et al., 2010 (link)). Paw and spinal cord slices were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody (1:200 in PBS, v/v, Millipore- DBA, Milan, Italy). Sections were rinsed with PBS and incubated with peroxidase-conjugated goat anti-rabbit IgG (1:2,000 Jackson Immuno Research, West Grove, PA, USA). Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). To authenticate the binding specificity for different antibodies, duplicate slices were incubated with only primary or secondary antibody; no positive staining was observed in these sections. Slices were quantitatively evaluated for a variance in immunoreactivity by computer-assisted color imaging (Leica QWin V3, Cambridge, UK). The percentage area of immunoreactivity (determined by the number of positive pixels) was expressed as percent of total tissue area (red staining). Replicates for all experimental conditions and histochemical staining were acquired from each rat in each experimental group. All analyses were carried out by two observers blinded to the treatment.

Pancreas and lung sections were incubated with the following primary antibodies: Anti-NRF2 (sc-365949, 1:200, Santa Cruz Biotechnology, CA, USA); anti-HO-1 (sc-136960, 1:200, Santa Cruz Biotechnology, CA, USA); anti-Mn-SOD (sc-137254, 1:200, Santa Cruz Biotechnology, CA, USA); anti-NLRP3 (sc-134306, 1:200, Santa Cruz Biotechnology, CA, USA); anti-Caspase-1 (sc-56036, 1:200, Santa Cruz Biotechnology, CA, USA); and anti-ASC (sc-514414, 1:200, Santa Cruz Biotechnology, CA, USA), as previously described [48 (link)]. Sections were then incubated with the following secondary antibodies: Peroxidase-conjugated bovine anti-mouse immunoglobulin G (IgG) or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA, USA). Specific marking was revealed with a biotin-conjugated goat anti-rabbit IgG or biotin-conjugated goat anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Graphic presentation of densitometric analyses was performed Image J software (v1.52a) as previously described [49 (link)]. All immunohistochemical analyses were conducted by an observer without knowledge of the treatments.