Arteries or cells were placed into RIPA lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein samples (30 μg) were separated with 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). The membrane was blocked with blocking buffer (TBS, 0.1% Tween-20, 5% non-fat milk or 2% BSA) for 2 hours at room temperature and incubated with primary antibodies against eNOS (1:1000, Cell Signaling Technology, Danvers, MA, USA), p-eNOS (ser1177) (1:1000, Abcam, Cambridge, MA, USA), arginase I (1:1000, Abcam, Cambridge, MA, USA), arginase II (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), UT-B (1:1000, a kindly gift from Dr. Trinh-Trang-Tan, INSERM, Paris, France), β-actin (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Goat anti-rabbit IgG horseradish peroxidase (1:10000, Abcam, Cambridge, MA, USA) or goat anti-mouse IgG horseradish peroxidase (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated for 60 minutes at room temperature the next day, respectively. The blots were developed with ECL kit (Applygen Technologies Inc, Beijing, China) and finally exposed to X-ray films. Relative protein expression levels were quantified by optical density analysis (Quantity-One software, Bio Rad Gel Doc 1000, Milan, Italy) and normalized to β-actin.
Goat anti rabbit igg horseradish peroxidase
Manufactured by Abcam
Sourced in United States
Goat anti-rabbit IgG horseradish peroxidase is a secondary antibody conjugated with horseradish peroxidase enzyme. It is used to detect and quantify the presence of rabbit primary antibodies in immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cox 2 rabbit polyclonal antibody, by Abcam (3 mentions)
Rabbit anti caspase 3 polyclonal antibody, by Abcam (3 mentions)
Anti parp rabbit polyclonal antibody, by Abcam (2 mentions)
Gel doc 1000, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Goat anti mouse igg horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
P enos ser1177, by Abcam (1 mentions)
Arginase 2, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
β actin, by Bio-Rad (1 mentions)
Ecl kit, by Applygen (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Dicamba, by Merck Group (1 mentions)
Chloramben, by Merck Group (1 mentions)
Picloram, by Merck Group (1 mentions)
Originpro 8, by OriginLab (1 mentions)
Clopyralid, by Merck Group (1 mentions)
8 protocols using goat anti rabbit igg horseradish peroxidase
1
Western Blot Analysis of Vascular Proteins
2
Lentivirus-induced Astrocyte Protein Analysis
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After being infected by lentivirus for 14 days, reactive astrocytes in the GFP and NEUROD1 groups were lysed using radioimmunoprecipitation assay buffer (containing both protease and phosphatase inhibitors) for 30 minutes at 4°C, and then centrifuged at 12,000 × g for 15 minutes. The supernatant was collected for gel analysis. Next, we separated the lysates using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 7.5% gel, and the lysates were then transferred onto a polyvinylidene difluoride membrane. Blocking was performed for 1 hour at room temperature with 10% fetal bovine serum in PBS. For the primary antibody incubation, antibodies including anti-casein kinase II subunit alpha (CSNK2A2; 1:1000; Invitrogen, Waltham, MA, USA; Cat# MA5-17062) and anti-pinin (PNN; 1:1000; Invitrogen; Cat# PA5-44563) were added to PBS with 0.05% Triton X-100, and the membrane was incubated overnight at 4°C. Next, the membrane was incubated with goat anti-rabbit IgG-horseradish peroxidase (1:2000; Abcam; Cat# ab6721) for 1 hour at 20°C. Signals were then detected on an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).
3
Hapten Synthesis and Analytical Characterization
4
Matrine Modulates NF-κB and MAPK Pathways
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The effects of matrine on NF-κB, MAPKs, and AKT pathways in RAW264.7 cells were evaluated by Western blotting. The RAW264.7 cells were seeded (2 × 106 cells/well) into 6-well plates and divided into 2 groups: cells treated with RANKL and cells treated with matrine (0 or 4 μM). Cells were evaluated by Western blotting at 0, 15, 30, 45, and 60 min to observe phosphorylation of IκB, P65, P50, ERK, JNK, P38, C-fos, and AKT. The 3 groups of RAW264.7 cells (control, treated with RANKL and matrine 0 or 4 μM) were cultured in 6-well plates (2 × 106 cells/well) for 7 d and measured by Western blotting. At d 7, the expression levels of osteoclastogenesis-related markers MMP-9, TRAP, cathepsin K, C-Src, and NFATc1 were determined. The primary antibodies included mouse anti-GAPDH and mouse anti–β-actin. Antibodies specific to P-ERK, ERK, P-JNK, JNK, P-P38, P38, P-C-fos, C-fos, P-AKT, AKT, P-IκB, IκB, P-P65, P65, P-P50, P50, MMP-9, cathepsin K, C-Src, TRAP, NFATc1, and β-actin were supplied by Abcam. Secondary antibodies included goat anti-rabbit IgG–horseradish peroxidase (Abcam) and donkey anti-goat horseradish peroxidase–HRP (Abcam).
5
Immunohistochemical Analysis of Apoptosis Markers
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For immunohistochemistry, some sections were incubated in goat normal serum (in order to block the nonspecific site), and subsequently with anti-caspase 3 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam, Cambridge, USA), anti-COX 2 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam, Cambridge, USA), and anti-PARP rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam, Cambridge, USA) overnight at 4 °C. The sections were washed with PBS and then incubated with secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit IgG, Abcam, Cambridge, USA) for 2 hours and detected by diaminobenzidine tetrahydrochloride for 5 minutes. Afterward, they were dehydrated and mounted. For negative controls, primary antibodies were omitted. For quantitative analysis, immunohistochemical photographs (n=5 photos from each sample were collected from all the mice in each experimental group) were assessed by densitometry using MacBiophotonics ImageJ 1.41a software on an ASUS personal computer. The data are expressed as a percentage of the total tissue area.
6
Immunohistochemical Analysis of Spinal Cord and DRG
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For immunohistochemistry, some sections were incubated with anti-caspase 3 rabbit polyclonal antibody (1:200 in PBS, v/v, Abcam), anti-COX 2 rabbit polyclonal antibody (1:200 in PBS, v/v, Abcam), and anti-S100ß rabbit polyclonal antibody (1:500 in PBS, v/v, Abcam) overnight at 4 °C. Sections were washed with PBS and incubated with secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit IgG, Abcam) for 2 h. For quantitative analysis, immunohistochemical photographs (n = five photos from each five-micrometer serial transverse sections of ipsilateral spinal cord segments of the sciatic nerve and related dorsal root ganglions, the thickness of between sampled sections was 48 μm for spinal cord and 36 μm for DRG) from all rats in each experimental group were assessed by densitometry using ImageJ software. Data are expressed as a percentage of total tissue area.
7
Immunohistochemical Analysis of Apoptosis and Inflammation Markers
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For immunohistochemistry, sections were incubated in normal serum (in order to block non-specific site), and then with anti-caspase 3 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam), anti-COX 2 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam) and anti-PARP rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam) overnight at 4 °C. Sections were washed with PBS and then incubated with secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit IgG, Abcam) for 2 h and detected by diaminobenzidine tetrahydrochloride for 5 min. After wards, they were dehydrated and mounted. For negative controls, primary antibodies were omitted. For quantitative analysis at percent of total tissue area, immunohistochemical photographs (n = 5 photos from each samples collected from all mice in each experimental group) were assessed by densitometry using MacBiophotonics ImageJ 1.41a software on an ASUS personal computer. Data are expressed as a percentage of total tissue area.
8
Immunohistochemical Evaluation of COX-2 Expression
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In order to evaluate the anti‐inflammatory activity of F. persica extract, cyclooxygenase‐2 (COX‐2) was taken by immunohistochemistry (Nasiry, Khalatbary, & Ebrahimzadeh, 2017 (link); Nasiry, Khalatbary, Ahmadvand, et al., 2017 (link)). After the rehydration tissue sections, they were incubated in goat normal serum (in order to block non‐specific site), and then with anti‐COX‐2 rabbit polyclonal antibody (1:100 in phosphate‐buffered saline (PBS), v/v, Abcam) overnight at 4°C. After washing with PBS, samples were incubated with secondary antibody conjugated with horseradish peroxidase (goat anti‐rabbit IgG, Abcam) and in order to detect the reactions, diaminobenzidine tetrahydrochloride was added for 5 min. Finally, they were dehydrated and mounted. To quantitatively analyze, five photographs of each sample (from all rats in each experimental group) were taken and assessed by densitometry using MacBiophotonics ImageJ software (National Institutes of Health). Data were expressed as percentages of total tissue area.
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