Pegylated human recombinant Arginase I cobalt [HuArgI (Co)-PEG5000] (Pegzilarginase) was a gift from Aeaglea BioTherapeutics (Aeaglea BioTherapeutics, Austin, TX, USA). The following primary antibodies were used in this study: Rabbit polyclonal anti-beta Actin, mouse monoclonal anti-MMP2[6E3F8], rabbit polyclonal anti-MMP9, rabbit polyclonal anti-ASS-1, and mouse monoclonal anti-vinculin [VIN-54] (Abcam Inc., Cambridge, UK). The following secondary antibodies were used in this study: Alexa Fluor 488 goat anti-mouse IgG (H + L) was obtained from Invitrogen). Anti-rabbit and anti-mouse HRP-conjugated secondary antibodies were obtained from Promega (Promega Co., Madison, WI, USA).
Rabbit polyclonal anti mmp 9
Manufactured by Abcam
Sourced in United Kingdom
Rabbit polyclonal anti-MMP-9 is an antibody that recognizes the matrix metalloproteinase-9 (MMP-9) protein. MMP-9 is an enzyme involved in the breakdown of extracellular matrix components. This antibody can be used to detect and study the expression of MMP-9 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Uorescence microscope, by Olympus (6 mentions)
Rabbit polyclonal anti caveolin 1, by Abcam (6 mentions)
Rabbit monoclonal anti occludin antibody, by Abcam (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Rabbit monoclonal anti cd31 antibody, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rabbit polyclonal anti beta actin, by Abcam (1 mentions)
Hrp conjugated anti rabbit and anti mouse secondary antibodies, by Promega (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Ripa lysis buffer, by ZSGB-BIO (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit monoclonal anti ppar γ, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti tgfβ, by Abcam (1 mentions)
Rabbit monoclonal anti il 6, by Cell Signaling Technology (1 mentions)
9 protocols using rabbit polyclonal anti mmp 9
1
Arginase I Cobalt Therapy: Antibody Analysis
2
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Zsbio) was used to extract protein from tissues and indicated cells. BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) was used to measure protein concentration. A total of 50 μg of protein was separated on 10% SDS-PAGE and blotted onto 0.22-μm nitrocellulose membranes. The membranes were blocked for 2 h with 5% nonfat dry milk diluted with tris-buffered saline (TBS) and incubated with primary antibodies [rabbit polyclonal anti-REST (1:100), rabbit polyclonal anti-MMP9 (1:100; Abcam), and mouse monoclonal anti-β-GAPDH (1:2,000; Abcam)] overnight at 4°C. The membranes were washed with TBS containing 0.1% Tween 20 (TBST) and then incubated with appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:2,000; goat anti-mouse, 1:2,000; Santa Cruz, Santa Cruz, CA, USA) for 1 h at 37°C. Enhanced chemiluminescence reagent (Merck Millipore, Darmstadt, Germany) was used to detect the signal on the membrane. The data were analyzed via densitometry using the Image-Pro plus software 6.0 (Bio-Rad, Hercules, CA, USA) and normalized to the expression of the internal control (GAPDH).
3
Comprehensive Protein Analysis Protocol
4
Quantitative Analysis of Cerebrovascular Markers
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At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4°C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), rabbit monoclonal anti-occludin antibody (1:100, Abcam), and rabbit monoclonal anti-CD31 antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan). We used the Image J software to perform the anlysis. Six images of slides were obtained per groups. Images were converted into an 8-bit format, and an intensity threshold was set and kept constant for all images analyzed. Mean uorescence intensities was calculated by dividing the uorescence intensities by the area of outlined regions.
5
Quantifying Protein Expression in Stroke
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At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4 °C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), and rabbit monoclonal anti-occludin antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan).
6
Quantifying Protein Expression in Stroke
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At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4 °C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), and rabbit monoclonal anti-occludin antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan).
7
Immunostaining of Brain Tissue Post-MCAO
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At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4°C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), rabbit monoclonal anti-occludin antibody (1:100, Abcam), and rabbit monoclonal anti-CD31 antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan).
8
Quantitative Analysis of Cerebrovascular Markers
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At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4°C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), rabbit monoclonal anti-occludin antibody (1:100, Abcam), and rabbit monoclonal anti-CD31 antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan). We used the Image J software to perform the anlysis. Six images of slides were obtained per groups. Images were converted into an 8-bit format, and an intensity threshold was set and kept constant for all images analyzed. Mean uorescence intensities was calculated by dividing the uorescence intensities by the area of outlined regions.
9
Immunostaining of Brain Tissue Post-MCAO
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At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4°C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), rabbit monoclonal anti-occludin antibody (1:100, Abcam), and rabbit monoclonal anti-CD31 antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan).
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