rabbit anti mouse mmp 9  (Proteintech)

Journal: Brain

Article Title: Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease

doi: 10.1093/brain/awz364

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Negative controls were processed using the same protocol with the omission of the primary antibody to assess non-specific labelling. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl * eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl * eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl * eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl * BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl * eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl * BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl * BioLegend 506308 Open in a separate window mAb = monoclonal antibody; pAb = polyclonal antibody.

Techniques: Flow Cytometry

Journal: Brain

Article Title: Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease

doi: 10.1093/brain/awz364

Figure Lengend Snippet: Cognitive preservation and restricted pathology following adoptive transfer of a CD115+ ACE10 monocyte subset in AD+ mice transgenic mice. (A–C) Schematic representation of experimental procedure and treatment groups. (A) CD115+ monocytes (MoBM) were isolated from the bone marrow of GFP+ donor mice and enriched by MACS microbeads and an anti-CD115 antibody column sorting procedure. (B) MoBM were then intravenously (i.v.) injected into the tail vein of AD+ recipient mice (n = 8 mice/group, all males). (C) Additional control groups included naïve wild-type (WT) mice and AD+ mice injected with PBS (n = 7 mice/group, all males). (D) Schematic timeline of the in vivo preclinical experiment. Pre-symptomatic AD+ mice exhibiting neuropathology at 8 months of age received monthly injections of 5–6 million GFP+MoWT or GFP+MoACE10 or PBS for 3 months (immunization regimen indicated by green arrows). At 11 months, mice underwent behavioural tests followed by tissue collection and analysis when 12 months of age. (E and F) Open field test in all AD+ treatment and naïve wild-type groups measuring: (E) ambulatory and (F) rearing activity. (G–K) Cognitive functions assessed by the Barnes maze test in both monocyte-treated groups as compared to the control PBS-injected group and naïve cognitively normal wild-type group (n = 6–8 mice/group). Incorrect entries (errors) for the following: (G) acquisition-training phase (Days 1–4), (H) memory retention (Day 7), (I) reversal phase (Days 8 or 9), and (J and K) memory test at Day 9. (J) Errors and (K) escape latency (s). (L) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in the hippocampus (HC), cingulate cortex (CC), and total brain in MoWT- and MoACE10-treated versus PBS-injected AD+ mice (n = 7–8 mice/group). (M) CAA score assessed as vascular Thio-S+ in AD+ mice (n = 6–8 mice/group). Data from an individual mouse as well as group mean ± SEM are shown. (N) Quantitative IHC analysis of hippocampal pre-(VGluT1+) and postsynaptic (PSD95+) areas in MoACE10-treated mice compared to PBS-injected control AD+ and naïve wild-type mice (n = 6–8 mice/group). Data from individual mice, lower and upper quartiles (as lower and upper horizontal lines in box), median (midline within box), and minimum/maximum values (whiskers), are shown. Percentage increase and decrease compared to control groups are shown in green. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. Significance between two-groups by unpaired two-tailed Student t-test. IR = immunoreactive.

Article Snippet: Negative controls were processed using the same protocol with the omission of the primary antibody to assess non-specific labelling. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl * eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl * eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl * eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl * BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl * eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl * BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl * BioLegend 506308 Open in a separate window mAb = monoclonal antibody; pAb = polyclonal antibody.

Techniques: Preserving, Adoptive Transfer Assay, Transgenic Assay, Isolation, Injection, Control, In Vivo, Activity Assay, Two Tailed Test

Journal: Stem Cells International

Article Title: Long Noncoding RNA SAMMSON Promotes Melanoma Progression by Inhibiting FOXA2 Expression

doi: 10.1155/2023/8934210

Figure Lengend Snippet: The sequences of all primers.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% BSA for 1 h at room temperature and incubated with anti-rabbit FOXA2 (1 : 1000; Cat. No. 8186; Cell Signaling Technology), anti-mouse MMP9 (1 : 1000; Cat. No. 13667; Cell Signaling Technology), anti-rabbit MMP2 (1 : 1000; Cat. No. 40994; Cell Signaling Technology), anti-rabbit GAPDH (1 : 5000; Cat. No.10494-1; Proteintech), and anti-mouse β -actin (1 : 10,000; Cat. No. A5441; Sigma-Aldrich) antibodies overnight at 4°C.

Techniques: Sequencing

Journal: Stem Cells International

Article Title: Long Noncoding RNA SAMMSON Promotes Melanoma Progression by Inhibiting FOXA2 Expression

doi: 10.1155/2023/8934210

Figure Lengend Snippet: SAMMSON silencing inhibits melanoma cell migration and invasion in vitro . (a, b) Analysis of the effect of SAMMSON knockdown on melanoma cell migration and invasion using Transwell assays. Representative images of migrated and invaded cells are shown in the top panel; quantitative results are shown below the images. (c–g) RT-qPCR analysis of the relative mRNA expression of E-cadherin, vimentin, N-cadherin, MMP9, and MMP2 in SAMMSON-silenced and control melanoma cells. (h, i) Western blotting analysis of the protein expression of MMP9 and MMP2 in SAMMSON-silenced and control melanoma cells. Scale bars = 100 μ m. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 vs. control cells.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% BSA for 1 h at room temperature and incubated with anti-rabbit FOXA2 (1 : 1000; Cat. No. 8186; Cell Signaling Technology), anti-mouse MMP9 (1 : 1000; Cat. No. 13667; Cell Signaling Technology), anti-rabbit MMP2 (1 : 1000; Cat. No. 40994; Cell Signaling Technology), anti-rabbit GAPDH (1 : 5000; Cat. No.10494-1; Proteintech), and anti-mouse β -actin (1 : 10,000; Cat. No. A5441; Sigma-Aldrich) antibodies overnight at 4°C.

Techniques: Migration, In Vitro, Knockdown, Quantitative RT-PCR, Expressing, Control, Western Blot

Journal: Stem Cells International

Article Title: Long Noncoding RNA SAMMSON Promotes Melanoma Progression by Inhibiting FOXA2 Expression

doi: 10.1155/2023/8934210

Figure Lengend Snippet: FOXA2 inhibits MMP9 transcription. (a, b) RT-qPCR and western blotting analyses of the mRNA and protein expression of MMP9 in NC-OE and FOXA2-OE melanoma cells. (c, d) RT-qPCR and western blotting analyses of the mRNA and protein expression of MMP9 in control and FOXA2-silenced melanoma cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 vs. control cells.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% BSA for 1 h at room temperature and incubated with anti-rabbit FOXA2 (1 : 1000; Cat. No. 8186; Cell Signaling Technology), anti-mouse MMP9 (1 : 1000; Cat. No. 13667; Cell Signaling Technology), anti-rabbit MMP2 (1 : 1000; Cat. No. 40994; Cell Signaling Technology), anti-rabbit GAPDH (1 : 5000; Cat. No.10494-1; Proteintech), and anti-mouse β -actin (1 : 10,000; Cat. No. A5441; Sigma-Aldrich) antibodies overnight at 4°C.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Nuclear Factor Kappa-B Signaling Is Integral to Ocular Neovascularization in Ischemia-Independent Microenvironment

doi: 10.1371/journal.pone.0101602

Figure Lengend Snippet: The mRNA levels for the genes; NFκB/p65 , α5 , β1 , ET-1 , MMP-9 , FAK and EPO , were quantified by Real time RT-PCR. Selected experiments, which measured the mRNA levels of NFκB/p65 , indicated that YC-1 and SN50 downregulated the mRNA levels of NFκB/p65 as compared to DMSO or SN50M-treated retinas, respectively. For the other genes listed above, the mRNA levels were upregulated in the DMSO-treated retinas and the rho/VEGF group that was left untreated. In contrast, retinas from YC-1-treated retinas exhibited a significant downregulation of the mRNA expression as compared to retinas that were treated with DMSO. Despite the sustained expression of VEGF, there were no detectable differences in the levels of CXCR4 and SDF-1 mRNA expression in the animals of all groups. ANOVA was used for statistical analyses. Mean ± SEM of mRNA level normalized to β-actin were calculated, [*** P <0.001 and ** P <0.01, as compared to respective controls]. Data are representative of 3 independent experiments. J. Sequence for the Primer Sets Used for the Quantitative Real-Time PCR Analysis .

Article Snippet: Rabbit polyclonal anti-mouse Matrix Metalloproteinase-9 (MMP-9) antibody was obtained from LifeSpan BioSciences (Seattle, WA).

Techniques: Quantitative RT-PCR, Expressing, Sequencing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Nuclear Factor Kappa-B Signaling Is Integral to Ocular Neovascularization in Ischemia-Independent Microenvironment

doi: 10.1371/journal.pone.0101602

Figure Lengend Snippet: Immunohistochemical analysis of NFκB/p65, FAK, α5β1, EPO, ET-1, and MMP-9, has indicated the expression levels of these proteins were significantly elevated in the rho/VEGF retinas that were left untreated. YC-1-treated retinas exhibited a significant decrease in the protein expression levels as compared with DMSO-treated retinas. Despite the sustained expression of VEGF, there were no detectable differences in the levels of CXCR4 and SDF-1 protein expression amongst the animals of all groups. Retinas were examined at 100× objective. Scale bar, 100 µm.

Article Snippet: Rabbit polyclonal anti-mouse Matrix Metalloproteinase-9 (MMP-9) antibody was obtained from LifeSpan BioSciences (Seattle, WA).

Techniques: Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: Nuclear Factor Kappa-B Signaling Is Integral to Ocular Neovascularization in Ischemia-Independent Microenvironment

doi: 10.1371/journal.pone.0101602

Figure Lengend Snippet: Immunohistochemical analysis of NFκB/p65, FAK, α5β1, EPO, ET-1, and MMP-9, has indicated the expression levels of these proteins were significantly elevated in the rho/VEGF retinas that were left untreated. YC-1-treated retinas exhibited a significant decrease in the protein expression levels as compared with DMSO-treated retinas. Despite the sustained expression of VEGF, there were no detectable differences in the levels of CXCR4 and SDF-1 protein expression amongst the animals of all groups. Retinas were examined at 100× objective. Scale bar, 100 µm.

Article Snippet: Rabbit polyclonal anti-mouse Matrix Metalloproteinase-9 (MMP-9) antibody was obtained from LifeSpan BioSciences (Seattle, WA).

Techniques: Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: Nuclear Factor Kappa-B Signaling Is Integral to Ocular Neovascularization in Ischemia-Independent Microenvironment

doi: 10.1371/journal.pone.0101602

Figure Lengend Snippet: Immunohistochemical analysis of NFκB/p65, FAK, α5β1, EPO, ET-1, and MMP-9, has indicated the expression levels of these proteins were significantly elevated in the rho/VEGF retinas that were left untreated. YC-1-treated retinas exhibited a significant decrease in the protein expression levels as compared with DMSO-treated retinas. Despite the sustained expression of VEGF, there were no detectable differences in the levels of CXCR4 and SDF-1 protein expression amongst the animals of all groups. Retinas were examined at 100× objective. Scale bar, 100 µm.

Article Snippet: Rabbit polyclonal anti-mouse Matrix Metalloproteinase-9 (MMP-9) antibody was obtained from LifeSpan BioSciences (Seattle, WA).

Techniques: Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: Nuclear Factor Kappa-B Signaling Is Integral to Ocular Neovascularization in Ischemia-Independent Microenvironment

doi: 10.1371/journal.pone.0101602

Figure Lengend Snippet: Immunohistochemical analysis of NFκB/p65, FAK, α5β1, EPO, ET-1, and MMP-9, has indicated the expression levels of these proteins were significantly elevated in the rho/VEGF retinas that were left untreated. YC-1-treated retinas exhibited a significant decrease in the protein expression levels as compared with DMSO-treated retinas. Despite the sustained expression of VEGF, there were no detectable differences in the levels of CXCR4 and SDF-1 protein expression amongst the animals of all groups. Retinas were examined at 100× objective. Scale bar, 100 µm.

Article Snippet: Rabbit polyclonal anti-mouse Matrix Metalloproteinase-9 (MMP-9) antibody was obtained from LifeSpan BioSciences (Seattle, WA).

Techniques: Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: Nuclear Factor Kappa-B Signaling Is Integral to Ocular Neovascularization in Ischemia-Independent Microenvironment

doi: 10.1371/journal.pone.0101602

Figure Lengend Snippet: NFκB/p65: Nuclear Factor Kappa B/P65 ; α5β1: Integrin Alpha-5 Beta-1 ; ET-1: Endothelin-1 ; MMP-9: Matrix Metalloproteinase-9 ; FAK: Focal Adhesion Kinase ; EPO: Erythropoietin.

Article Snippet: Rabbit polyclonal anti-mouse Matrix Metalloproteinase-9 (MMP-9) antibody was obtained from LifeSpan BioSciences (Seattle, WA).

Techniques:

Journal: Circulation research

Article Title: Matrix Metalloproteinase-9 Is Required for Adequate Angiogenic Revascularization of Ischemic Tissues

doi: 10.1161/01.RES.0000111527.42357.62

Figure Lengend Snippet: MMP-9 expression and activity are induced at early time points in hindlimb muscle tissues exposed to ischemia. MMP-9 protein expression was assayed in the nonischemic (day 0) or ischemic (1, 3, 7, and 14 days) WT adductor muscle tissue lysates using SDS-PAGE gelatin zymography (top) and Western blotting (middle). Both indicate early induction of MMP-9 (day 1), with levels peaking at 3 days and returning to baseline by 7 days after ischemia (latent “Pro” MMP-9 and activated MMP-9). Quantification of total gelatinolytic activity associated with MMP-9 or MMP-2 (both pro- and activated bands) in WT and MMP-9−/− tissue lysates, normalized using standards (“MMP Stds”) (n=4 animal for each time point), illustrates the significant early induction of MMP-9 in response to ischemia in the WT, and similar induction of MMP-2 in the WT and MMP-9−/−. *P<0.05 vs 0 day time point. For a detailed separate analysis of MMP-2 and MMP-9, see online Figure 1.

Article Snippet: MMP-9 was identified using a rabbit anti-mouse MMP-9 antibody (Chemicon) and RRX-donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Activity Assay, SDS Page, Zymography, Western Blot

Journal: Circulation research

Article Title: Matrix Metalloproteinase-9 Is Required for Adequate Angiogenic Revascularization of Ischemic Tissues

doi: 10.1161/01.RES.0000111527.42357.62

Figure Lengend Snippet: Capillary histochemical affinity staining indicates lack of an ischemia-induced angiogenic response in MMP-9−/− mice. Examination of tissue cross-sections stained with endothelial specific Griffonia simplicifolia lectin (green) indicates that capillary density increases in WT, but not in MMP-9−/− tissues in response to ischemia (scale bar=20 μm). All nuclei counterstained with Hoechst (blue). Insets illustrate in addition BrdU-positive (red) immunodetection of proliferating cells (pink, due to localization to nuclei counterstained in blue) with capillary staining (green), as well as a negative control with no primary antibody (“No 10”). Proliferating cells were found only in ischemic WT tissues supporting the presence of a robust angiogenic response. Quantification of capillary density displays a significant increase in capillary number within WT but not within MMP-9−/− ischemic tissues (n=4 for each point, *P<0.05 vs MMP-9−/−), supporting MMP-9 role in ischemia-induced angiogenic response.

Article Snippet: MMP-9 was identified using a rabbit anti-mouse MMP-9 antibody (Chemicon) and RRX-donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Techniques: Staining, Immunodetection, Negative Control

Journal: Circulation research

Article Title: Matrix Metalloproteinase-9 Is Required for Adequate Angiogenic Revascularization of Ischemic Tissues

doi: 10.1161/01.RES.0000111527.42357.62

Figure Lengend Snippet: MMP-9 deficiency decreases restoration of microvascular perfusion capacity in response to ischemia. Time course of perfusion capacity imaged on whole-mount tissue specimens using fluorescence microangiography (scale bars=200 μm). Nonischemic muscle contains numerous perfused capillaries with no apparent differences between WT and MMP-9−/− mice. The number of perfused capillaries drops dramatically after femoral artery ligation, illustrated at day 3 in both WT and in MMP-9−/− tissues, in spite of existing anatomical structures (illustrated by specific staining in Figure 2). Perfusion capacity is significantly better restored in WT tissues compared with the MMP-9−/− tissues (day 7), and actually becomes higher than the baseline at day 14, whereas it remains deficient in MMP-9−/− tissue, supporting the role of MMP-9 for proper revascularization. In addition, note the increased number of points of capillary intersections, indicative of branching, and increased tortuosity in WT compared with MMP-9−/− ischemic tissue (day 14) or compared with nonischemic tissues. Vascular perfusion capacity of adductor muscle (left graph), quantified as fluorescence extracted from tissue homogenates (n=4 animals per point), dropped dramatically after femoral artery ligation in both WT and MMP-9−/− tissue. By 14 days, WT vascular perfusion capacity surpassed initial capacity, indicating robust revascularization and angiogenesis, whereas in the MMP-9−/− ischemic tissues, perfusion capacity remained lower than initial capacity (*P<0.05 vs WT 14 day), illustrating blunted revascularization. Quantification of capillary branching density (from confocal microscope images of tissues, n=4 animals for each point) indicated a significant increase in branching density in the WT after 14 days compared with normal nonischemic tissues (*P<0.05 vs nonischemic). On the other hand, MMP-9−/− ischemic tissues had a decreased density of branches at 14 days (*P<0.05 vs WT), suggesting that lack of capillary branching contributed to decreased perfusion capacity.

Article Snippet: MMP-9 was identified using a rabbit anti-mouse MMP-9 antibody (Chemicon) and RRX-donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Techniques: Fluorescence, Ligation, Staining, Microscopy

Journal: Circulation research

Article Title: Matrix Metalloproteinase-9 Is Required for Adequate Angiogenic Revascularization of Ischemic Tissues

doi: 10.1161/01.RES.0000111527.42357.62

Figure Lengend Snippet: Detection of capillaries, MMP-9 promoter activity and protein, as well as macrophages suggests a role for macrophage MMP-9 in the early angiogenic response to ischemia. Left, Visualization by fluorescence microangiography of perfused capillaries (green) and MMP-9 promoter activity (red) in the GelB/LacZ Tg mice in nonischemic and ischemic tissue (day 3), indicates a perivascular localization of cells actively transcribing MMP-9 (arrows) in ischemic tissues. Inset, Typical finding of MMP-9 promoter activity at branching points. Middle, Positive detection for macrophages (red) in ischemic WT tissues was frequently observed at points of capillary (green) intersections. On the other hand, no macrophages were detected in MMP-9−/− ischemic tissues. Inset, Negative control for staining with no primary antibody (No 10). All nuclei are counterstained with Hoechst (blue). Right, Double immunohistochemistry illustrates colocalization (arrows) of (top) macrophages (green) and MMP-9 promoter activity (red, GelB/LacZ Tg mouse), as well as (bottom) macrophages (green) and MMP-9 protein (red, WT mouse). Insets demonstrate the specificity of immunostaining, ie, consecutive sections processes without primary antibody (No 10) and the lack of MMP-9–positive signal in ischemic tissues harvested from the MMP-9−/− mice. All scale bars=20 μm. Taken together, the results of these analyses suggest a link between MMP-9, macrophages, and angiogenic branching in ischemic tissues.

Article Snippet: MMP-9 was identified using a rabbit anti-mouse MMP-9 antibody (Chemicon) and RRX-donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay, Fluorescence, Negative Control, Staining, Immunohistochemistry, Immunostaining

Journal: Circulation research

Article Title: Matrix Metalloproteinase-9 Is Required for Adequate Angiogenic Revascularization of Ischemic Tissues

doi: 10.1161/01.RES.0000111527.42357.62

Figure Lengend Snippet: Transplantation with WT bone marrow rescues ischemia-induced angiogenic capillary branching in the MMP-9−/− mice. Nonischemic tissues illustrate similar levels of capillary perfusion capacity in the WT or MMP-9−/−. Left, Fluorescence microangiography. Middle, Increased perfusion capacity was detected in WT ischemic tissues, whereas MMP-9−/− ischemic tissues showed an impaired perfusion (day 14), consistent with inadequate angiogenesis. Transplantation (Tx) of WT bone marrow to MMP-9−/− mice (right) rescued perfusion capacity, as suggested by the high capillary tortuosity and branching in the MMP-9−/− ischemic tissue (top right). All scale bars=100 μm. Bottom right, MMP-9 (red)–positive macrophages (green) were detected (arrows) at early time points in the MMP-9−/− ischemic tissues of mice transplanted with WT marrow by double immunohistochemistry (scale bar=20 μm). Inset, Demonstration that macrophages (green) are bone marrow–derived because they are positive for the donor rare allele CD45.1 (red). All nuclei are counterstained with Hoechst (blue). Graph, Quantification of capillary branching by fluorescence microangiography confirms increased branching in WT (solid bar) ischemic tissue (P<0.05 vs nonischemic) and decreased branching in MMP-9−/− (white bar) ischemic tissue (P<0.05 vs nonischemic) and demonstrates that transplantation (Tx) of WT bone marrow into MMP-9−/− mice (striped) increases capillary branching in MMP-9−/− ischemic tissues vs MMP-9−/− nonischemic tissues (*P<0.05), as well as ischemic tissues of nontransplanted MMP-9−/− (*P<0.05), bringing it to levels comparable to WT ischemic tissues (P=NS).

Article Snippet: MMP-9 was identified using a rabbit anti-mouse MMP-9 antibody (Chemicon) and RRX-donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Techniques: Transplantation Assay, Fluorescence, Immunohistochemistry, Derivative Assay