Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (A)–(F), n = 3 individual mice/group. (B) Volcano plot from RNA-seq identifying differentially expressed genes (DEGs), with at least log 2 4-fold change at FDR < 0.05, between virus-induced lung and splenic CD4 + T cells. (C) Induced genes from (B) ranked by fold induction. (D) Heatmap of gene expression induction of all induced enzymes (orange) with Arg1 position indicated (red). (E and F) Representative (E) RNA-seq tracks of the Arg1 locus and (F) Arg1 expression (RNA-seq) in splenic and lung CD4 + T cells after influenza infection. (G) Experimental setup for (H)–(K). (H) Representative fluorescence-activated cell sorting (FACS) plots of ARG1 protein expression in WT and Arg1 CKO mice. (I) Viral titers in the lung at days 7 and 9 p.i. Representative experiment (of two independent experiments) shown with n = 4–5 mice per group per time point. (J) Representative hematoxylin and eosin (H&E) histology staining and (K) pathology score of the lungs at day 9 p.i. Combined data from two independent experiments, n = 11–13. *p < 0.05 (two-tailed Student’s t test). Each dot represents a sample from an individual mouse. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: RNA Sequencing Assay, Virus, Expressing, Infection, Fluorescence, FACS, Staining, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (B)–(L). (B and C) Numbers of (B) total lung mononuclear cells and (C) lung CD4 + T cells at days 7 and 9 p.i., n = 12–17. (D and E) Representative FACS plots showing (D) influenza-specific (NP311–325 tetramer + ) lung CD4 + T cells and their CD11a and CD49d expression (in red) and (E) percentages of lung CD11a + CD49d + cells. (F–H) Numbers of lung (F) CD11a + CD49d + CD4 + T cells, (G) Ki67 + CD4 + T cells, and (H) NP311–235 tetramer + CD4 + T cells at days 7 and 9 p.i. in WT and Arg1 CKO mice, n = 4–6. (I and J) Representative FACS plots of (I) lung NP311–235 tetramer + CD4 + T cells and (J) T-bet expression by CD11a lo CD49d lo , CD11a + CD49d + , and NP311–235 tetramer + CD4 + T cells at day 9 p.i. (K and L) Numbers of (K) intracellular IFN-γ + , IL-2 + , and IL-10 + -producing lung CD4 + T cells (at day 7 p.i. and after in vitro restimulation with PR8-infected or non-infected dendritic cells), with (L) a representative FACS plot of data in (K), n = 14–15. *p < 0.05, **p < 0.01. (B and C) Kruskal-Wallis test; (F–I and L) Mann-Whitney test. ns, no statistically significant difference. (B, C, K, and L) Combined data from three individual experiments. (F–H) One representative of three independent experiments shown. Each dot represents a sample from an individual mouse. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Expressing, In Vitro, Infection, MANN-WHITNEY

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (B)–(D). (B) Representative FACS plot showing transferred WT and Arg1 CKO CD4 + T cells. (C and D) Numbers of CD4 + T cells recovered from (C) spleens and (D) lymph nodes 7 days post transfer, n = 4. (E) Experimental setup for (F)–(H). (F) Representative FACS plot of lung WT and Arg1 CKO CD11a + CD49d + cells at day 7 p.i. (G and H) Frequency of lung (G) CD11a + CD49d + CD4 + T cells and (H) Ki67 + CD4 + T cells at day 7 p.i., n = 5. (I) Experimental setup for (J)–(R). (J) Body weight of mice receiving no cells, n = 1, or WT or Arg1 CKO cells, n = 8–10. (K) Percentage of CD4 + T cells in the spleens, n = 8–10. (L and M) Numbers of CD4 + T cells in (L) spleens and (M) colons, n = 8–10. Representative of two individual experiments. (N and O) Weight of (N) spleens and (O) colons of mice injected with WT or Arg1 CKO CD4 + T cells, n = 15. (P–R) Colon pathology of mice injected with WT or Arg1 CKO based on (P) severity and (Q) inflammation, assessed via H&E histology staining. (R) Representative H&E staining of the colons. (N–Q) Combined data from two individual experiments, n = 15 individual mice/group. Each dot represents a sample from a single mouse. *p < 0.05, **p < 0.01, ***p < 0.001. (C, D, G, and H) paired Student’s t test; (J–Q) two-tailed Student’s t test.

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Injection, Staining, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (B)–(G). (B) Splenic naive CD4 + T cells in uninfected WT and Arg1 CKO animals, n = 14–16 (data from four combined experiments shown). (C and D) Representative (C) histogram of cell trace violet dilution at day 3 post activation and (D) division index (n = 3–4, one representative of two individual experiments shown). (E) 5-ethynyl-2′-deoxyuridine (EdU) incorporation at days 2.5 (n = 7–8) and 5 (n = 5) post activation (data from two combined individual experiments). (F) IFN-γ and IL-10 secretion at day 3 post activation (n = 5–7, data from two combined individual experiments shown). (G) Representative FACS plots showing intracellular IFN-γ and IL-10 staining at 5 days post activation. (H) Simplified schematic of ARG1 and ARG2 subcellular localization to cytoplasm (cyto) or mitochondria (mito). (I) Splenic naive CD4 + T cells in naive WT and Arg2 KO mice, n = 12–14 (data derived from four individual experiments). (J and K) CD4 + T cells from WT and Arg2 KO mice were activated in vitro for 3 days and (J) IFN-γ, IL-10, and (K) IL-17A measured (IFN-γ, n = 3; IL-10, n = 6, data from two combined individual experiments; IL-17A, n = 3–4). (L and M) RNA-seq analyses of CD4 + T cells from WT, Arg1 C KO, or global Arg2 KO mice at 22 h post in vitro activation (n = 3 individual mice/group) with (L) numbers of differentially expressed genes (DEGs) and (M) venn diagram and list of overlapping enriched biological pathways derived from DEGs. Each dot represents a sample from a single mouse. *p < 0.05, **p < 0.01. (D–F and I–K) Two-tailed Student’s t test. ns, no statistically significant difference. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Activation Assay, Staining, Derivative Assay, In Vitro, RNA Sequencing Assay, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Simplified diagram of the classical arginase pathway. (B–D) CD4 + T cells were isolated from the spleens of WT and Arg1 CKO mice and stimulated in vitro with anti-CD3 and anti-CD28 antibodies for 22–24 h. (B) Ornithine, n = 3 samples from individual mice, done in triplicate; (C) polyamine, n = 3 samples from individual mice; and (D) arginine abundancy in the CD4 + T cells, as determined by liquid chromatography-mass spectrometry, n = 3 samples from individual mice. (E–G) (E) Glycolysis (ECAR) and oxidative phosphorylation (OCR), as determined by Seahorse metabolomic profiling in response to oligomycin (oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (fccp), and rotenone (rot) are shown with (F) accompanying statistical evaluation. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. (E–G) n = 4, each dot represents individual mouse, one representative of three total experiments shown. *p < 0.05. (B, D, F, and G) Mann-Whitney test; (C) Kruskal-Wallis test. ns, no statistically significant difference. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Isolation, In Vitro, Liquid Chromatography, Mass Spectrometry, MANN-WHITNEY

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A and B) Volcano plot of (A) differential metabolite abundance in in vitro -activated (22–24 h) CD4 + T cells from WT and Arg1 CKO mice, n = 4–6. Positive and negative ionization mode features (gray), unannotated features that exceeded log 2 FC > 0.26 (~20% change) and adjusted p value < 0.05 (black), annotated features which exceeded log 2 FC > 0.26 and adjusted p value < 0.05 (red- and blue-highlighted and labeled for clarity), and (B) glutamine abundancy. (C) Simplified schematic of intersecting arginine and glutamine pathways. (D and E) Abundance of (D) glutamate and (E) α-ketoglutarate (αKG). For αKG, n = 3 individual mice shown with technical replicates. (F) Schematic of Gpt2 activity in glutamine metabolism. (G and H) Representative FACS plots showing glutamate pyruvate transaminase 2 (GPT2) protein expression in WT, (G) Arg1 KO, and (H) Arg2 KO CD4 + T cells on day 3 post in vitro activation. (I) Percentage of IFN-γ + , IL-10 + , and IFN-γ-IL-10 double-positive T cells assessed via flow cytometry after in vitro stimulation with or without AOA treatment, n = 2–4 (representative of two independent experiments). *p < 0.05, **p < 0.01, ****p < 0.0001. (B, D, E, G, and I) Two-tailed Student’s t test; (L) one-way ANOVA. ns, no statistically significant difference, TCA cycle, tricarboxylic acid cycle. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: In Vitro, Labeling, Activity Assay, Expressing, Activation Assay, Flow Cytometry, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A and B) Representative FACS plots showing (A) arginase 1 (ARG1) expression and (B) CAT-1 expression at days 2 and 3 in healthy donor CD4 + T cells after in vitro stimulation, n = 4. (C) Amount of IFN-γ (left), IL-10 (middle), or ratio of IL-10 to IFN-γ (IL-10/IFN-γ) (right) secreted by CD4 + T cells after CD3+CD46 stimulation in vitro for 36 h in the presence of Nω-hydroxy-nor-L-arginine (nor-NOHA) or vehicle, n = 4. (D–K) CD4 + T cells were isolated from the blood of patients with arginase-1 deficiency (designated as P) and age-matched healthy controls (designated as HC). (D–F) IFN-γ and IL-10 secretion by CD3+CD46-activated CD4 + T cells (36 h, primary activation) with (D) individual values and (E) cumulative data for the IL-10/IFN-γ ratio during primary activation, n = 4 (four individual patients with multiple blood samples taken over a 2-year period), and (F) after CD3+CD46 restimulation for ~18–20 h post resting (5 days), n = 4 (individual patients, some with multiple blood samples taken over a 2-year period). (G and H) 5-ethynyl-2′-deoxyuridine (EdU) incorporation at day 5 post primary stimulation with (G) a representative FACS plot and (H) cumulative data, n = 2 healthy controls with technical duplicates, and n = 3 patients. (I) Percent of live CD4 + T cells after restimulation, as described under (F), n = 2 healthy controls done in duplicate and n = 3 patients, 1 with a technical duplicate. (J) Polyamine abundancy in resting CD4 + T cells. (K) Glycolysis (ECAR) and oxidative phosphorylation (OCR) as determined by Seahorse metabolomic profiling of CD4 + T cells after CD3+CD46 stimulation for 24 h. (L–O) ARG1 was over expressed in isolated CD4 + T cells from three healthy donors (n = 3) by electroporation of ARG1 into the cells with (L) a representative FACS plot showing ARG1 expression in control (CTRL) electroporated versus ARG1 electroporated CD4 + T cells prior to activation. ARG1 overexpressing or control CD4 + T cells were activated in vitro for 36 h. (M) Representative FACS plot, (N) cumulative data showing IFN-γ and IL-10 production, and (O) percent of cells that are Ki67 + . (P) Pathway analysis of DEGs derived from microarray analyses of CD3+CD46-activated CD4 + T cells (6 h) from patients 1 and 2 and two age-matched healthy control cells, n = 2. *p < 0.05, **p < 0.01. (C, N, and O) Paired Student’s t test; (E, F, H, and I) Mann-Whitney test. fccp, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; oligo, oligomycin; rot, rotenone. See also and .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Expressing, In Vitro, Isolation, Activation Assay, Electroporation, Control, Derivative Assay, Microarray, MANN-WHITNEY

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Virus, Isolation, Recombinant, Staining, Cell Isolation, Flow Cytometry, RNA Sequencing Assay, In Vitro, Microarray, CRISPR, Control, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Progranulin induces immune escape in breast cancer via up-regulating PD-L1 expression on tumor-associated macrophages (TAMs) and promoting CD8 + T cell exclusion

doi: 10.1186/s13046-020-01786-6

Figure Lengend Snippet: PGRN promotes CD8 + T cell exclusion and inhibits tumor immunity. a . The expression of CD4, CD8 and Granzyme B in WT and PGRN KO mice breast cancer tissue sections was tested by immunohistochemical staining. b . Colocalization of CK19 (red), F4/80 (red), CD206 (red), PD-L1 (red) and CD8 (green) in WT and PGRN KO mice breast cancer tissue sections was examined by immunofluorescence analysis, and the nucleus was stained with DAPI (blue). c-d WT and PGRN −/− peritoneal macrophages were co-cultured with mouse spleen lymphocytes activated by αCD3/CD28. Here CM stands for control medium, without αCD3/CD28 stimulation. c . Flow cytometry was used to measure the proportion of activated CD8 + T cells (IFN-γ + CD8 + T cells) and (D) Ki-67 + CD8 + T cells. * p < 0.05; ** p < 0.01

Article Snippet: The slides were incubated overnight at 4 °C with the following antibodies for multicolor immunofluorescence staining: F4/80 (Cell Signaling Technology, 30,325 T, 1:200), PD-L1 (Abcam, ab213480, 1:100), CD206 (proteintech, 18,704–1-AP, 1:200), Arg1 (Abcam, ab96183, 1:100), iNOS (Abcam, ab178945, 1:200), CD8 (Bioss, bs0648R, 1:100), CD4 (Bioss, bs-0647R, 1:100), CK19 (Abcam, ab52625, 1:200), and PD-1 (Abcam, ab214421, 1:100).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Cell Culture, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Progranulin induces immune escape in breast cancer via up-regulating PD-L1 expression on tumor-associated macrophages (TAMs) and promoting CD8 + T cell exclusion

doi: 10.1186/s13046-020-01786-6

Figure Lengend Snippet: The interaction of PD-1/PD-L1 mediates the immunosuppressive function of PGRN in breast cancer. a . Expression of PD-1 in WT and PGRN KO mice breast cancer tissue sections was detected with immunohistochemical staining. b . PD-L1 (green), CD4 (green), CD8 (green) expression differences and co-localization with PD-1 (red) in WT and PGRN KO mice breast cancer tissue sections were examined by immunofluorescence, and the nucleus was stained with DAPI (blue). c . Mouse splenic lymphocytes activated with or without αCD3/CD28 antibody were co-cultured with WT or PGRN −/− peritoneal macrophages, and the frequency of PD-1 + CD8 + T cells was tested by flow cytometry. d-e Wild-type peritoneal macrophages were co-cultured with splenic lymphocytes preactivated by αCD3/CD28 antibody, and then anti-PD-1 or anti-PD-L1 neutralizing antibodies were added or not to the co-culture system. d . CD8 + T cell activation and ( e ) CD8 + T cell proliferation were detected by flow cytometry. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The slides were incubated overnight at 4 °C with the following antibodies for multicolor immunofluorescence staining: F4/80 (Cell Signaling Technology, 30,325 T, 1:200), PD-L1 (Abcam, ab213480, 1:100), CD206 (proteintech, 18,704–1-AP, 1:200), Arg1 (Abcam, ab96183, 1:100), iNOS (Abcam, ab178945, 1:200), CD8 (Bioss, bs0648R, 1:100), CD4 (Bioss, bs-0647R, 1:100), CK19 (Abcam, ab52625, 1:200), and PD-1 (Abcam, ab214421, 1:100).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Cell Culture, Flow Cytometry, Co-Culture Assay, Activation Assay

Journal: Molecular cell

Article Title: Deciphering functional tumor-immune crosstalk through highly multiplexed imaging and deep visual proteomics.

doi: 10.1016/j.molcel.2024.12.023

Figure Lengend Snippet: Figure 3. T cell subsets have distinctive spatial proteome signatures in colorectal tumor microenvironment (A and B) Ranking of protein quantified in cytotoxic T cells (CTLs) (A) and helper T cells (TH) (B), ranked by median transformed intensity values detected by mass spectrometry, with cell-type-specific markers highlighted. (C and D) Volcano plots illustrating proteomic comparisons of CTLs (C) and TH (D) between the lamina propria and tumor epithelial regions, with T cell-activity- related markers highlighted. Significantly enriched proteins are denoted as yellow or green dots (two-sided t test, false discovery rate [FDR] < 0.05, S0 = 0.1, n = 3). (E and F) Gene Ontology (GO) term analysis of significantly altered proteins in CTLs (E) and TH (F) residing in the lamina propria and tumor epithelial regions.

Article Snippet: For CD4 + ARG1 staining, the sections were incubated overnight at 4 C with primary antibodies (anti-CD4-FITC, 1:100, R&D FAB8165G; anti-ARG1-PE, 1:200,Miltenyi Biotec 130-127-839) in a humidified chamber.

Techniques: Transformation Assay, Mass Spectrometry, Activity Assay

Journal: Molecular cell

Article Title: Deciphering functional tumor-immune crosstalk through highly multiplexed imaging and deep visual proteomics.

doi: 10.1016/j.molcel.2024.12.023

Figure Lengend Snippet: Figure 5. Cytotoxic T cells (CTLs) exhibit close proximity to tumors with a suppressive impact on tumor proliferation (A) Density gradient in the background indicates distance to tumor islands, with brighter yellow representing greater distance from tumor cells. Left panel only has the gradient whereas the middle, zoomed panel additionally depicts yellow masks of the CTLs and the right panel red dots of helper T cells (TH), respectively. (B) Representative images showcasing markers for T cell phenotyping. (C) T cell gating based on the expression of CD3, CD8, CD45RA, CD45RO, and PD1 to analyze the corresponding T cell subpopulations. (D) Representative images showing CD3- and CD8-positive CTLs in tumor parenchyma (TIL-CTLs) with absent CD45RA and CD45RO expression. (E) Distance heatmap depicting proximity and interactions between tumor cells and T and B lymphocyte subtypes, with lower values indicating higher proximity as in Figure 4E. (F) Histogram of Ki67/Ki67+ tumor cell number as a function of distance from RARO CTLs. Data represent three technical replicates. Statistical significance was determined using one-way ANOVA. ***p < 0.001, **p < 0.01. (G) Representative image displaying masks of tumor cells at varying proximities to RARO TIL-CTLs.

Article Snippet: For CD4 + ARG1 staining, the sections were incubated overnight at 4 C with primary antibodies (anti-CD4-FITC, 1:100, R&D FAB8165G; anti-ARG1-PE, 1:200,Miltenyi Biotec 130-127-839) in a humidified chamber.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Sensory and Sympathetic Nervous System on Fracture Healing in Ovariectomized Mice

doi: 10.3390/ijms21020405

Figure Lengend Snippet: Macrophage subtype profile during the early fracture healing phase. Macrophages in the callus tissue of WT, Tac1−/−, α-CGRP−/−, and SYX mice 5 days after fracture were detected using F4/80 as an overall macrophage marker ( A ), inducible nitric oxide synthase (iNos) as a marker for M1 macrophages ( B ), and Arginase 1 (Arg1) as a marker for M2 macrophages ( C ). Values are calculated as a percentage of total callus cells and are shown as median ± Min/Max. ( D ) Representative images of the F4/80, iNos, and Arg1 staining in the callus tissue of a Tac1−/− mouse. Red boxes demonstrate the enlarged view. Red arrow points to stained cells. Magnification: 20× (TissueFAXSi plus). B = bone, C = Callus tissue, BM = bone marrow, F = Fracture site, M=Muscle. * = p ≤ 0.05, ** = p ≤ 0.01. N = 5.

Article Snippet: Then, the following primary antibodies were incubated over night at 4 °C: CD 4—1:100 (# 25229, Cell Signaling Technology (CST), Danvers, MA, USA), CD 8—1:400 (# 98941, CST), F4/80—1:250 (# 70076, CST), iNos—1:75 (# 13120, CST), Arg1—1:100 (# 93668, CST) or the appropriate concentration of the isotype control (# ab172730, Abcam, Cambridge, UK) in antibody diluent (# 8112, SignalStain ® Antibody Diluent, CST).

Techniques: Marker, Staining