primary antibodies against m1  (Novus Biologicals)

Journal: British journal of pharmacology

Article Title: Activation of M 4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

doi: 10.1111/bph.16392

Figure Lengend Snippet: FIGURE 1 Outline of the experimental design. Study 1 evaluated the impact of systemic xanomeline (XAN) on behavioural responses related to TS and comorbid disorders. Study 2 was designed to evaluate the involvement of M4 receptors in the effects of XAN, leveraging the selective M4 antagonist, VU06028418 and the M4 positive allosteric modulator (PAM), VU0467154. Study 3 was designed to evaluate the involvement of M1 receptors in the effects of XAN, using the selective M1 antagonist, VU0255035 and cevimeline (CEV), a muscarinic agonist with high affinity and potency on M1 and M3 but not M4 receptors. Study 4 evaluated the involvement of the dorsal striatum in the behavioural effects of XAN. Study 5 was used for the neurochemical investigations. The timelines for treatments and procedures are outlined in each panel. Abbreviations: PPI, prepulse inhibition of the acoustic startle. WB, western blotting. IF, immunofluorescence. For further details, see text.

Article Snippet: Primary antibodies against M1 (Cat# NBP1–87466, RRID: AB_11021120, dilution 1:750; Novus Biological, Littleton, CO), M4 (Cat# NBP3-03052, RRID:AB_2943415, dilution 1:1000; Novus Biological) were incubated in TBS-T containing 3% (w/v) BSA buffer overnight at 4 C. Next, blots were washed in TBS-T and then incubated in TBS-T containing goat anti-rabbit HRP-conjugated (Cat# 31462, RRID: AB_228338, dilution 1:10000; ThermoFisher Scientific, Waltham, MA) secondary antibodies, for 90 min at room temperature.

Techniques: Inhibition, Western Blot, Immunofluorescence

Journal: British journal of pharmacology

Article Title: Activation of M 4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

doi: 10.1111/bph.16392

Figure Lengend Snippet: FIGURE 5 The M1-selective antagonist VU0255035 (3–30 mgkg1, PO) does not reverse the ameliorative effects of xanomeline (XAN; 5 mgkg1, IP) in TS-related behaviours. Panels A-D show the combined effects of VU0255035 and XAN on (a) frequency of body and head jerks, (b) overall duration of grooming stereotypies, (c) acoustic startle amplitude and (d) acoustic prepulse inhibition (PPI) in CIN-d and control (Ctrl) mice. Panels (e)–(h) represent the effects of VU0255035 and XAN on the same behavioural paradigms in D1CT-7 and wild type (WT) male littermates. Data were analysed with two-way ANOVAs. X, P < 0.05 for main effect comparison of vehicle (VEH) with XAN; *, P < 0.05 for comparisons of vehicle-treated D1CT- 7 versus WT or CIN-d versus Ctrl mice; O, P < 0.05 for comparison of XAN and VU0244035 (30 mgkg1) versus VEH and VU0244035 (30 mgkg1). All data are shown as means ± SEM. n = 8 per group. For further details, see text.

Article Snippet: Primary antibodies against M1 (Cat# NBP1–87466, RRID: AB_11021120, dilution 1:750; Novus Biological, Littleton, CO), M4 (Cat# NBP3-03052, RRID:AB_2943415, dilution 1:1000; Novus Biological) were incubated in TBS-T containing 3% (w/v) BSA buffer overnight at 4 C. Next, blots were washed in TBS-T and then incubated in TBS-T containing goat anti-rabbit HRP-conjugated (Cat# 31462, RRID: AB_228338, dilution 1:10000; ThermoFisher Scientific, Waltham, MA) secondary antibodies, for 90 min at room temperature.

Techniques: Inhibition, Control, Comparison

Journal: British journal of pharmacology

Article Title: Activation of M 4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

doi: 10.1111/bph.16392

Figure Lengend Snippet: FIGURE 9 CIN-d mice show a significant reduction in M4, but not M1, receptor levels in the dorsal striatum. Representative immunoblot (a) and quantification of M1 (b) and M4 (c) expression levels in dorsal striatum expressed as a percentage of the control group (Ctrl). Values are expressed as mean ± SEM, n = 6.

Article Snippet: Primary antibodies against M1 (Cat# NBP1–87466, RRID: AB_11021120, dilution 1:750; Novus Biological, Littleton, CO), M4 (Cat# NBP3-03052, RRID:AB_2943415, dilution 1:1000; Novus Biological) were incubated in TBS-T containing 3% (w/v) BSA buffer overnight at 4 C. Next, blots were washed in TBS-T and then incubated in TBS-T containing goat anti-rabbit HRP-conjugated (Cat# 31462, RRID: AB_228338, dilution 1:10000; ThermoFisher Scientific, Waltham, MA) secondary antibodies, for 90 min at room temperature.

Techniques: Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: A High Red Blood Cell Distribution Width Predicts Failure of Arteriovenous Fistula

doi: 10.1371/journal.pone.0036482

Figure Lengend Snippet: Leukocytes, defined as CD45-positive cells, were found in the radial artery or cephalic vein or both in 39% of the patients. Monocytes/macrophages, defined as CD68-positive cells, were found in the vessel wall in 37% of the patients. Both cell types were most frequently located in the arterial and venous intima and media. The larger squares are magnifications of the smaller ones.

Article Snippet: After sequential incubation with 3% hydrogen peroxide (Merck, Darmstadt, Germany) for 15 minutes at room temperature to block endogenous peroxidase, with avidin and biotin (DakoCytomation, Glostrop, Denmark) for 20 minutes at room temperature, and with Fc-receptor blocker for 30 minutes (Innovex Biosciences, Richmond, CA), the slides were incubated with primary antibodies against CD68 (IgG3, clone: PG-M1), CD45 (IgG1, clone: 2B11+PD7/26), and CD31 (All from DakoCytomation, Glostrop, Denmark) overnight at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: A High Red Blood Cell Distribution Width Predicts Failure of Arteriovenous Fistula

doi: 10.1371/journal.pone.0036482

Figure Lengend Snippet: A–C, Patients in whom CD68-positive cells were found in the radial artery or cephalic vein wall had significantly higher WBCs ( p = 0.002) (B), but there were no significant differences in RDW (A) or the number of monocytes (C). D–F, No significant differences in levels of RDW (D), WBC (E), or number of monocytes (F) were found in patients whose radial artery or cephalic vein wall contained CD45-positive cells.

Article Snippet: After sequential incubation with 3% hydrogen peroxide (Merck, Darmstadt, Germany) for 15 minutes at room temperature to block endogenous peroxidase, with avidin and biotin (DakoCytomation, Glostrop, Denmark) for 20 minutes at room temperature, and with Fc-receptor blocker for 30 minutes (Innovex Biosciences, Richmond, CA), the slides were incubated with primary antibodies against CD68 (IgG3, clone: PG-M1), CD45 (IgG1, clone: 2B11+PD7/26), and CD31 (All from DakoCytomation, Glostrop, Denmark) overnight at 4°C.

Techniques:

Journal: The EMBO Journal

Article Title: Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single‐cell profiling

doi: 10.15252/embj.2020105123

Figure Lengend Snippet: Gating strategy of eye macrophages. CD11b + or CD45 + cells underwent doublet exclusion by FSC‐W, ‐A, and ‐H gating and subsequent dead cell exclusion (fixable viability dye, FVD). Retinal microglia were specified as CD45 lo CD11b + , macrophages in the ciliary body and cornea as CD45 lo CD11b + CD64 + F4/80 + . Gating strategy of blood monocytes. Leukocytes underwent doublet exclusion by FSC‐W, ‐A, and ‐H gating and subsequent dead cell exclusion (fixable viability dye, FVD). CD45 + CD11b + myeloid blood cells were further subdivided in CD45 + CD11b + CD115 + Ly6C hi and CD45 + CD11b + CD115 + Ly6C lo monocytes.

Article Snippet: Cells were stained with primary antibodies directed against CD11b (M1/70), CD45 (30‐F11), CD115 (AFS98), F4/80 (BM8) (eBioscience), CD64 (X54‐5/7.1), Ly6C (AL‐21), and Ly6G (1A8) at 4°C for 20 min at a concentration of 1:200 if not indicated differently.

Techniques:

Journal: The EMBO Journal

Article Title: Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single‐cell profiling

doi: 10.15252/embj.2020105123

Figure Lengend Snippet: Left: Creation of Acta1 GFP/+ :Acta1 +/+ bone marrow chimeras. Right: Donor‐derived GFP + CD45 lo CD11b + were detectable in the recipient retina by flow cytometry 20 weeks after bone marrow reconstitution. FACS Plots are representative for five animals from one experiment. Quantification of GFP + cells among CD45 + CD11b + retinal microglia by flow cytometry. Data are presented as mean ± s.e.m. One symbol represents one mouse. Typical retinal flat mount from Acta1 GFP/+ :Acta1 +/+ bone marrow chimeras 20 weeks after reconstitution. Donor‐derived GFP + Iba1 + cells (arrow) and GFP − Iba1 + resident microglia (asterisks) are shown. Pictures are representative for three animals from one experiment. Scale bars represent 50 µm. Microscopy‐based quantification of GFP + Iba1 + retinal microglia in the inner (IPL) and outer plexiform layer (OPL). One symbol represents one mouse. Data are presented as mean ± s.e.m. Scheme of a fate mapping experiment using Cx3cr1 CreERT2 :Rosa26‐YFP female mice. Tamoxifen (TAM) and progesterone injection were performed at embryonic day 9.0 (E9.0). Mice were subsequently evaluated at postnatal day 0 (P0). Administration of TAM leads to intra‐embryonic excision of a stop sequence flanked by loxP sites (gray triangles) in Cx3cr1 expressing cells which causes stable and steady YFP expression under the control of the Rosa26 promotor. Direct fluorescence microscopic visualization for YFP (green), the macrophage marker Iba1 (red) and DAPI for the nuclei (blue) at P0. YFP + Iba1 + double‐positive cells are marked by arrows. YFP − Iba1 + single‐positive cells are labeled by asterisks. Representative images out of five examined animals are shown. Scale bars represent 25 µm. Quantitative analysis of regional YFP expression in Iba1 + macrophages in TAM‐induced and untreated Cx3cr1 CreERT2 :Rosa26‐YFP mice. Bars represent means ± s.e.m. Quantification was done from three (untreated) or five (TAM) mice obtained from one (untreated) or two (TAM) independent experiments. Level of significance determined by Mann–Whitney test between TAM and untreated revealed * P < 0.05 and Kruskal–Wallis test between retina, ciliary body, and cornea revealed * P = 0.0204.

Article Snippet: Cells were stained with primary antibodies directed against CD11b (M1/70), CD45 (30‐F11), CD115 (AFS98), F4/80 (BM8) (eBioscience), CD64 (X54‐5/7.1), Ly6C (AL‐21), and Ly6G (1A8) at 4°C for 20 min at a concentration of 1:200 if not indicated differently.

Techniques: Derivative Assay, Flow Cytometry, Microscopy, Injection, Sequencing, Expressing, Control, Fluorescence, Marker, Labeling, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single‐cell profiling

doi: 10.15252/embj.2020105123

Figure Lengend Snippet: Scheme of a fate mapping experiment using adult Cx3cr1 CreERT2 :Rosa26‐YFP mice. Tamoxifen (TAM) injection was performed at postnatal day 42 (P42). Mice were evaluated at 2, 12, and 24 weeks post‐injections. Administration of TAM leads to the excision of a stop sequence flanked by loxP sites (gray triangles) in Cx3cr1 expressing cells in the eye which causes stable YFP expression under the control of the Rosa26 promotor. Flow cytometric measurement of the persistence of YFP + retinal microglia (rMG), cliliary body (cb) MΦ, and corneal (c) MΦ in adult Cx3cr1 CreERT2 :Rosa26‐YFP mice. Doublets and dead cells were excluded by FSC‐W and viability dye. Representative flow cytometry plots from two independent experiments with at least six mice are displayed. Kinetics of YFP labeling in macrophages of the healthy eye. Symbols represent means ± s.e.m. rMG are shown as squares (2 weeks: n = 10 mice, 12 weeks: n = 9 mice, 24 weeks: n = 12 mice, Kruskal–Wallis ns P > 0.05), cbMΦ are depicted as triangles (2 weeks: n = 4 samples from eight mice, 12 weeks: n = 3 samples from six mice, 24 weeks: n = 6 samples from twelve mice, Kruskal–Wallis ns P > 0.05) and cMΦ as circles (2 weeks: n = 6 mice, 12 weeks: n = 8 mice, 24 weeks: n = 12 mice, one‐way ANOVA *** P < 0.0001). Data were obtained from two (cMΦ: 2 weeks, cbMΦ: 2 and 12 weeks), three (rMG: 2 weeks, cbMΦ: 24 weeks), or four (rMG: 12 and 24 weeks, coMΦ: 12 and 24 weeks) independent experiments. Sketch of the Flt3 ‐dependent Cre‐mediated recombination system with excision of the loxP‐flanked stop‐sequences leading to expression of YFP under the control of the Rosa26 promotor in Flt3 Cre :Rosa26‐YFP mice. Left: Representative flow cytometric characterization of rMG by CD45 and CD11b and cbMΦ and cMΦ by CD45, CD11b, CD64, and F4/80 in Flt3 Cre :Rosa26‐YFP mice. Doublets and dead cell were excluded. Right: representative flow cytometric images depicting YFP expression in eye tissue macrophages of 12‐ or 52‐week‐old Flt3 Cre :Rosa26‐YFP mice. Typical images were taken from two independent experiments with six to seven mice. Quantification of the percentage of YFP + eye macrophages at 12 and 52 weeks of age. rMG are shown as squares (12 weeks: n = 7 mice, 52 weeks: n = 6 mice), cbMΦ as triangles (12 weeks: n = 3 samples from six mice, 52 weeks: n = 3 samples from six mice), and cMΦ as circles (12 weeks: n = 7 mice, 52 weeks: n = 6 mice). Data are presented as means ± s.e.m. and were acquired in two independent experiments.

Article Snippet: Cells were stained with primary antibodies directed against CD11b (M1/70), CD45 (30‐F11), CD115 (AFS98), F4/80 (BM8) (eBioscience), CD64 (X54‐5/7.1), Ly6C (AL‐21), and Ly6G (1A8) at 4°C for 20 min at a concentration of 1:200 if not indicated differently.

Techniques: Injection, Sequencing, Expressing, Control, Flow Cytometry, Labeling

Journal: The EMBO Journal

Article Title: Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single‐cell profiling

doi: 10.15252/embj.2020105123

Figure Lengend Snippet: A Left, Experimental setup of surgically connected parabiotic mice. Acta1 GFP/+ and Acta 1 +/+ mice underwent parabiosis for 2, 12, and 20 weeks before analysis. Right, quantification of GFP + Iba1 + microglia in the retina (rMG, squares, n.d. = not detectable), ciliary body (cbMΦ, triangles, Mann–Whitney ns P > 0.05), and cornea (cMΦ, circles, Kruskal–Wallis ** P = 0.0024) of parabiotic mice. Blood chimerism of CD45 + CD11b + Ly6C hi GFP + cells in the analyzed wild‐type mice was 37.7 ± 3.2% (2 weeks), 27.5 ± 2.7% (12 weeks), and 34.7 ± 3.7% (20 weeks). Symbols represent mean ± s.e.m. of three (2 weeks), four (12 weeks) or five (20 weeks) individual mice. Scale bars represent 50 µm. B–D Representative immunofluorescence images from the retina (20 weeks), ciliary body (12 weeks), and cornea (20 weeks) from flat mounts of Acta1 +/+ parabiotic mice. GFP + Iba1 + double‐positive cells are marked by arrows, GFP − Iba1 + single‐positive cells are labeled by asterisks and GFP + Iba1 − leukocytes are indicated by arrow heads. Pictures are representative of three animals. E Flow cytometric quantification of RFP + cells in Ccr2‐RFP mice among CD45 + CD11b + CD115 − Ly6C int granulocytes (triangles, n = 4), CD45 + CD11b + CD115 + Ly6C lo monocytes (filled squares, n = 4 mice), CD45 + CD11b + CD115 + Ly6C hi monocytes (open squares, n = 4 mice), CD45 lo CD11b + rMG (squares, n = 6 mice), CD45 + CD11b + CD64 + F4/80 + cbMΦ (triangles, n = 6 samples from 12 mice), and CD45 + CD11b + CD64 + F4/80 + cMΦ (filled circles, n = 12 mice, unpaired t ‐test *** P < 0.0001). Data were obtained from one (rMG) or two independent experiments (blood, cbMΦ, cMΦ). Data are presented as means ± s.e.m. F–H Flow cytometry of eye macrophages from healthy Ccr2 RFP/+ mice (left) and representative histograms (right, Ccr2 RFP/+ solid red line, Ccr2 +/+ controls dotted gray line). I Flow cytometry of myeloid blood cells from Ccr2 RFP/+ mice. Left: CD45 + CD11b + cells were further subdivided according to the expression of Ly6C and CD115 into CD45 + CD11b + CD115 − Ly6C int granulocytes (gate 1), CD45 + CD11b + CD115 + Ly6C hi inflammatory (gate 2), and CD45 + CD11b + CD115 + Ly6C lo resident monocytes (gate 3), respectively. Right: representative histograms are shown ( Ccr2 RFP/+ solid red line, Ccr2 +/+ controls dotted gray line). Four mice were investigated. J Top left, sketch of Ccr2‐RFP construct. Top right, typical confocal picture for CCR2 (red), Iba1 (green), and collagen IV (Coll IV, white) revealing Ccr2‐RFP expression in a blood vessel (arrow, left image, scale bar represents 20 µm) and no RFP signal in retinal microglia (right images, scale bar represents 100 µm). Bottom, representative pictures of the ciliary body and cornea immunolabeled with F4/80 (green), CCR2 (red), and DAPI (blue). Arrows indicate RFP + F4/80 + cells. Asterisks point to RFP − F4/80 + cells. Representative images from two independent experiments with three mice are displayed. Scale bars represents 20 µm.

Article Snippet: Cells were stained with primary antibodies directed against CD11b (M1/70), CD45 (30‐F11), CD115 (AFS98), F4/80 (BM8) (eBioscience), CD64 (X54‐5/7.1), Ly6C (AL‐21), and Ly6G (1A8) at 4°C for 20 min at a concentration of 1:200 if not indicated differently.

Techniques: MANN-WHITNEY, Immunofluorescence, Labeling, Flow Cytometry, Expressing, Construct, Immunolabeling

Journal: The EMBO Journal

Article Title: Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single‐cell profiling

doi: 10.15252/embj.2020105123

Figure Lengend Snippet:

Article Snippet: Cells were stained with primary antibodies directed against CD11b (M1/70), CD45 (30‐F11), CD115 (AFS98), F4/80 (BM8) (eBioscience), CD64 (X54‐5/7.1), Ly6C (AL‐21), and Ly6G (1A8) at 4°C for 20 min at a concentration of 1:200 if not indicated differently.

Techniques: Blocking Assay, Sequencing, Library Quantification, RNA HS Assay, Sensitive Assay, Software, Irradiation, Fluorescence, Microscopy, Imaging

Journal: Journal of clinical urology

Article Title: Characterization of the Macrophage Infiltrate in a Case of Xanthogranulomatous Pyelonephritis

doi: 10.1177/2051415817716014

Figure Lengend Snippet: Immunohistochemical staining of renal parenchyma affected by xanthogranulomatous pyelonephritis demonstrates positivity for the M1 macrophage marker iNOS and secreted cytokine IFN-γ. Cells in the nearby normal renal tissue are negative for these markers.

Article Snippet: In brief, tissues were stained with primary antibodies against the M1 markers iNOS (Thermo Fisher Rockford, IL 1:300) and IFN-γ (Abcam Cambridge, MA 1:600) as well as the M2 markers CD163 (Abcam Cambridge, MA 1:600) and CD206 (LSBIO, Seattle, WA 1:400).

Techniques: Immunohistochemical staining, Staining, Marker