rat anti mac2 antibody  (Cedarlane)

Journal: Scientific Reports

Article Title: Finerenone ameliorates diabetic kidney disease exacerbated by deletion of natriuretic peptide/guanylyl cyclase-A signaling and dietary high-protein load

doi: 10.1038/s41598-025-25362-0

Figure Lengend Snippet: Podocyte damage and inflammatory changes are worsened by HPD in systemic GC-A knockout mice. ( a ) Immunohistochemical (IHC) staining for WT-1 in glomerulus. The graph shows the number of WT-1-positive cells per glomerulus as quantitative analysis. ( b ) IF staining for nephrin in each group. Scale Bar represents 50 μm. ( c ) Representative images of electron microscopic analysis in each group. The graphs indicate quantitative analysis of foot process width and GBM thickness. Scale Bar represents 1 μm. ( d ) IHC staining of MAC2 in glomerulus (▶). The graph shows the number of MAC2-positive macrophages per glomerulus. Scale Bar represents 50 μm. ( e , f ) Glomerular mRNA expression levels of Tgfb1 , Ctgf , Serpine1 , Ccl2 , Emr1 , Icam1 , and Vcam1 in each group. ( g ) Renal mRNA expression levels of Agt , Ren1 , and Sgk1 in each group. β-Actin ( Actb ) was used as internal control. Values are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant.

Article Snippet: Primary antibodies used for immunohistochemical and immunofluorescence studies were anti-GC-A antibody (GTX109810, GeneTex, Inc., Irvine, CA, USA), anti-MAC2 antibody (CL8942F, Cedarlane, Ontario, Canada), anti-Wilms tumor 1 (WT1) antibody (sc-15421, Santa Cruz Biotechnology, Dallas, TX), anti-nephrin antibody (AF3159, R&D Systems, Inc., Minneapolis, MN, USA), and anti-Tie2 antibody (AF762, R&D Systems, Inc.).

Techniques: Knock-Out, Immunohistochemical staining, Immunohistochemistry, Staining, Expressing, Control

Journal: Scientific Reports

Article Title: Finerenone ameliorates diabetic kidney disease exacerbated by deletion of natriuretic peptide/guanylyl cyclase-A signaling and dietary high-protein load

doi: 10.1038/s41598-025-25362-0

Figure Lengend Snippet: Podocyte damage and profibrotic changes are worsened by HPD in endothelial cell-specific GC-A knockout mice. ( a ) IHC staining of WT-1 in glomerulus. The graph shows the number of WT-1-positive cells per glomerulus as quantitative analysis. Scale Bar represents 50 μm. ( b ) IF staining for nephrin. Scale Bar represents 50 μm. ( c ) Representative images of electron microscopic analysis in each group. The graphs indicate quantitative analysis of foot process width and GBM thickness. Scale Bar represents 1 μm. ( d ) IHC staining of MAC2 in glomerulus (▶). The graph shows the number of MAC2-positive macrophages per glomerulus. Scale Bar represents 100 μm. ( e ) Glomerular mRNA expression levels of Tgfb1 , Ctgf , Fn1 , Serpine1 , Ccl2 , and Emr1 in each group. ( f ) Renal mRNA levels of Fn1 , Serpine1 , Lcn2 , Agt, and Sgk1 in each group. β-Actin ( Actb ) was used as internal control. Values are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant.

Article Snippet: Primary antibodies used for immunohistochemical and immunofluorescence studies were anti-GC-A antibody (GTX109810, GeneTex, Inc., Irvine, CA, USA), anti-MAC2 antibody (CL8942F, Cedarlane, Ontario, Canada), anti-Wilms tumor 1 (WT1) antibody (sc-15421, Santa Cruz Biotechnology, Dallas, TX), anti-nephrin antibody (AF3159, R&D Systems, Inc., Minneapolis, MN, USA), and anti-Tie2 antibody (AF762, R&D Systems, Inc.).

Techniques: Knock-Out, Immunohistochemistry, Staining, Expressing, Control

Journal: Circulation Research

Article Title: Macrophage DNases Limit Neutrophil Extracellular Trap–Mediated Defective Efferocytosis in Atherosclerosis

doi: 10.1161/CIRCRESAHA.125.326353

Figure Lengend Snippet: DNase deficiency in hematopoietic cells impairs neutrophil extracellular trap (NET) clearance in atherosclerotic plaques. A , The relative levels of DNase1 and DNase1L3 in lesional macrophages (Mac2+), smooth muscle cells (sm-actin [smooth muscle actin]+), and endothelial cells (CD31+) were quantified by measuring DNase1 and DNase1L3 fluorescence intensity in antibody-labeled aortic root sections of 16-week Western diet–fed Ldlr −/− mice. B , Bone marrow–derived macrophages (BMDMs) from wild-type (WT) and Dnase1 −/− Dnase1l3 −/− (double knockout [DKO]) mice were exposed to NETs (250 ng/mL) for either 2 or 6 hours. The quantity of remaining NETs in the supernatant was measured by Picogreen assay, and the NET clearance efficiency was determined relative to input NET concentration. C , BMDMs from WT and DKO mice were incubated for 2 hours with pHrodo-red labeled NETs. After washes, fluorescence microscopy was performed, and the percentage of macrophages that showed engulfment of fluorescent NETs was quantified. D , Eight-week-old female Ldlr −/− mice were lethally irradiated followed by administration of bone marrow cells from either WT or DKO mice. Six weeks postbone marrow reconstitution, WT and hematopoietic cell–specific DNase1/DNase1L3 knockout (DNase-HCKO) mice were fed a Western-type diet for 16 weeks and then euthanized for analysis. E , Brachiocephalic artery from WT and DNase-HCKO mice were homogenized, and the tissue extract was analyzed for DNase activity by single radial enzyme diffusion assay. n=10 mice per group. F , Aortic tree from both groups of mice (n=5) were harvested, fragmented, and maintained in culture as explants, followed by exposure to vehicle or NETs for 4 hours. The supernatant was collected for measurement of ( G ) DNase activity and ( H ) NET clearance efficiency. I , Lesional NET level was quantified by immunostaining for MPO (myeloperoxidase) and citrullinated histone H3 (Cit-H3) in DAPI (4′,6-diamidino-2-phenylindole)–stained aortic root sections of WT and DNase-HCKO mice. n=10 mice per group. Scale bar, 50 µm. J , Hematoxylin and eosin–stained aortic root sections were analyzed for total atherosclerotic lesion area, ( K ) necrotic area, and ( L ) plaque necrosis as a percentage of total lesion area. The regions of plaque necrosis are demarcated by the black dashed line. n=10 to 11 mice per group. Scale bar, 50 µm. M , In situ efferocytosis assay in aortic root sections labeled with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) reagent to detect apoptotic cells (ACs; red) followed by Mac2 immunolabelling (green) to identify lesional macrophages. Lesional efferocytosis efficiency was calculated as the ratio of macrophage-associated ACs to free-lying ACs. White arrows indicate ACs associated with macrophages, whereas white arrowheads show free-lying TUNEL+ cells. n=10 mice per group. Scale bar, 25 µm. N , Quantification of lesional collagen content in Mason’s trichrome-stained aortic root sections. n=10 mice per group. Scale bar, 50 µm. All data are represented as mean±SEM. Test for normality was conducted by the Shapiro-Wilk test. P value was determined by the Mann-Whitney U test ( B , C , E , G , and H ), or unpaired t test ( I through N ). AU indicates arbitrary units; Mφ, macrophage; ND, not detected; and S.N., supernatant.

Article Snippet: After TUNEL labeling, the sections were immunostained with anti-Mac2 (galectin-3) antibody as described above and counterstained with DAPI.

Techniques: Fluorescence, Labeling, Western Blot, Derivative Assay, Double Knockout, Picogreen Assay, Concentration Assay, Incubation, Microscopy, Irradiation, Knock-Out, Activity Assay, Diffusion-based Assay, Immunostaining, Staining, In Situ, TUNEL Assay, MANN-WHITNEY

Journal: Circulation Research

Article Title: Macrophage DNases Limit Neutrophil Extracellular Trap–Mediated Defective Efferocytosis in Atherosclerosis

doi: 10.1161/CIRCRESAHA.125.326353

Figure Lengend Snippet: Neutrophil extracellular traps (NETs) cleave MerTK to impair efferocytosis. A , bone marrow–derived macrophages (BMDMs) were exposed to NETs (250 ng/mL), then incubated with cytochalasin D (1 µmol/L) for 30 minutes, followed by the addition of fluorescently labeled apoptotic cells (ACs). After washes, AC-binding efficiency was quantified by fluorescence microscopy. n=5 biological replicates. B , Quantification of cell surface MerTK levels in BMDMs treated with or without NETs for 2 hours. n=5 biological replicates. C , ELISA-based measurement of soluble MerTK in the cell culture supernatants of BMDMs exposed for 2 hours with indicated concentrations of NETs. n=3 biological replicates. D , Quantification of soluble MerTK in the cell culture supernatant of wild-type (WT) and double knockout (DKO) BMDMs exposed to vehicle or NETs (250 ng/mL) for 2 hours. n=4 biological replicates. E , Quantification of MerTK levels in lesional macrophages (Mac2+) by immunostaining of aortic root sections of 16-week Western diet–fed bone marrow chimeric WT and hematopoietic cell–specific DNase1/DNase1L3 knockout (DNase-HCKO) Ldlr −/− mice. n=10 mice per group. F , ELISA-based analysis of soluble MerTK levels in the peritoneal lavage of bone marrow chimeric WT and DNase-HCKO mice 2 hours after intraperitoneal injection of NETs without or with DNase1. n=5 mice per group. G , As mentioned above, except that WT and DNase-HCKO mice were adoptively transferred either WT or DKO macrophages intraperitoneally 16 hours before injection of NETs. n=5 mice per group. H , Eight-week-old male C57BL/6J mice were lethally irradiated and injected with bone marrow cells from either WT or MerTK cleavage–resistant (MerTK CR ) male mice. Six weeks later, bone marrow chimeric mice were injected with NETs (1 µg IP) and fluorescently labeled ACs 2 hours later. Peritoneal lavage was performed 1 hour later for analysis of ( I ) soluble MerTK by ELISA; ( J ) macrophage MerTK expression by flow cytometry, and ( K ) macrophage efferocytosis efficiency by flow cytometry. n=5 mice per group. The data are represented as mean±SEM. Data were tested for normal distribution using the Shapiro-Wilk test. P values were calculated using the Mann-Whitney U test ( A , B , E , I through K ) and the Kruskal-Wallis test with Dunn multiple comparisons correction ( D , F , and G ). Mφ indicates macrophage; and MFI, median fluorescence intensity.

Article Snippet: After TUNEL labeling, the sections were immunostained with anti-Mac2 (galectin-3) antibody as described above and counterstained with DAPI.

Techniques: Derivative Assay, Incubation, Labeling, Binding Assay, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Cell Culture, Double Knockout, Immunostaining, Western Blot, Knock-Out, Injection, Irradiation, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: Circulation Research

Article Title: Macrophage DNases Limit Neutrophil Extracellular Trap–Mediated Defective Efferocytosis in Atherosclerosis

doi: 10.1161/CIRCRESAHA.125.326353

Figure Lengend Snippet: Integrated stress response inhibitor (ISRIB) enhances vascular DNase activity and neutrophil extracellular trap (NET) clearance in murine atherosclerosis. A , Ten-week-old female Ldlr −/− mice were fed a Western diet (WD) for 16 weeks. During the final 4 weeks, one group of mice received ISRIB (1 mg/kg IP) daily, whereas the other group received vehicle. B , Aortic explants from vehicle and ISRIB-treated mice were exposed to NETs for measurement of DNase activity, and ( C ) NET clearance efficiency, in the supernatants. D , Aortic root sections were immunostained with anti-MPO (myeloperoxidase) and anticitrullinated histone H3 (CitH3) antibody for quantification of lesional NET levels, or ( E ) MerTK levels in lesional Mac2+ macrophages. F , Aortic root sections were labeled with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL; red) and immunostained with anti-Mac2 antibody (green) for measurement of lesional in situ efferocytosis efficiency as a ratio of macrophage-associated apoptotic cells (ACs):free-lying ACs. White arrows indicate TUNEL+ ACs associated with a macrophage. White arrowheads show free-lying TUNEL+ cells. G , Hematoxylin and eosin–stained aortic root sections were used for quantification of total lesion area, and ( H ) necrotic area, in vehicle and ISRIB-treated mice. The necrotic regions are demarcated by the red dashed lines. I , Quantitative polymerase chain reaction–based analysis of Tnf , Il1b , and Il6 , in vascular tissues obtained from vehicle and ISRIB-treated mice. n=10 mice per group. The data are represented as mean±SEM. Data were tested for normal distribution using Shapiro-Wilk test. P values were calculated using unpaired t test ( B through F , and H ), Mann-Whitney U test ( G and I ). AC indicates apoptotic cell; MFI, median fluorescence intensity; Mφ, macrophage; and ND, not detected.

Article Snippet: After TUNEL labeling, the sections were immunostained with anti-Mac2 (galectin-3) antibody as described above and counterstained with DAPI.

Techniques: Activity Assay, Western Blot, Labeling, TUNEL Assay, In Situ, Staining, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Fluorescence