Journal: Science Advances

Article Title: A flexible electronic strain sensor for the real-time monitoring of tumor regression

doi: 10.1126/sciadv.abn6550

Figure Lengend Snippet: ( A to P ) Immunohistochemistry of tumors excised from mice treated for 6 days with vehicle (vehicle), treated for 5 hours with erlotinib, or treated for 6 days with erlotinib. Stains are for cleaved caspase 3 (CC3), a marker associated with cell death; Ki67, a marker associated with cell proliferation; EGFR; and phosphorylated EGFR (pEGFR). Erlotinib is an active inhibitor of EGFR and prevents phosphorylation. ( Q to V ) Hematoxylin and eosin stains of (Q and R) tumors and (U and V) skin from mice that did or did not wear FAST for 6 days. In the histological sampling, there is no noticeable difference in the cell shapes or distributions between the samples from mice wearing the sensor and the samples from mice not wearing the sensor. Scale bars, 100 μm.

Article Snippet: Immunohistochemistry staining used the following antibodies: EGFR (D38B1) XP Rabbit mAb (#4267, Cell Signaling Technology, Danvers, USA), Phospho-EGFR (Tyr 1068 ) (D7A5) XP Rabbit mAb (#3777, Cell Signaling Technology), Cleaved Caspase-3 (Asp 175 ) (D3E9) Rabbit mAb (#9579, Cell Signaling Technology), Ki67 Polyclonal Antibody (#27309-1-AP, Proteintech Group, Rosemont, USA), and Biotinylated Goat Anti-Rabbit IgG (H+L) (ab64256, Abcam, Cambridge, UK).

Techniques: Immunohistochemistry, Marker, Phospho-proteomics, Sampling

Journal: Science Advances

Article Title: A flexible electronic strain sensor for the real-time monitoring of tumor regression

doi: 10.1126/sciadv.abn6550

Figure Lengend Snippet: ( A to D ) FAST reads out tumor volume progression or regression continuously at 5-min intervals in Balb/c mice with subcutaneous A20 B cell lymphoma tumors receiving 40 μg of CpG and 4 μg of anti-OX40 ( n = 5) or vehicle ( n = 4) treatments over (A and B) the first few hours following treatment or (C and D) the entire treatment period in the same mice. Individual mouse sensor trend lines are presented as seven-point moving averages. ( E and F ) Tumor volume measurements using calipers confirm FAST readouts over the entire treatment period. ( G to L ) Immunohistochemistry of tumors excised from mice treated for 6 days with vehicle (vehicle) or treated once with CpG + anti-OX40 (treated). Staining is against (G and H) CC3, (I and J) Ki67, and (K and L) OX40. The treated stains are from tumors excised within 6 hours after treatment initiation. T + , CpG and anti-OX40 treatment; T − , vehicle treatment; S + , with FAST sensor; S − , no FAST sensor. Scale bars, 50 μm. Data are presented as individual data point or curves. Bold line, average; (B) unpaired two-tailed Student’s t test; (D and F) one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Immunohistochemistry staining used the following antibodies: EGFR (D38B1) XP Rabbit mAb (#4267, Cell Signaling Technology, Danvers, USA), Phospho-EGFR (Tyr 1068 ) (D7A5) XP Rabbit mAb (#3777, Cell Signaling Technology), Cleaved Caspase-3 (Asp 175 ) (D3E9) Rabbit mAb (#9579, Cell Signaling Technology), Ki67 Polyclonal Antibody (#27309-1-AP, Proteintech Group, Rosemont, USA), and Biotinylated Goat Anti-Rabbit IgG (H+L) (ab64256, Abcam, Cambridge, UK).

Techniques: Immunohistochemistry, Staining, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner

doi: 10.1186/s12964-024-01535-8

Figure Lengend Snippet: Hyperactivity of CD73/adenosine axis promotes myocardial metabolic shift in a sex-dependent manner during HH and protein expression of the other ectonucleotidases. A - B Representative Immunofluorescence and graphical presentation of the co-staining of F4/80 and Glut1 with nuclei (DAPI). Color channels were adjusted in the merged images to enhance the visualization of all the respective fluorescence dyes. Scale bar, 50 μm. C - E Representative Oil Red O (ORO) and Periodic Acid Schiff (PAS) staining of myocardial sections and their respective graphical presentations showing lipid and glycogen deposition percentages ( n = 4–6 sections per 4–6 mice per group) Scale bar, 50 μm. F - H Representative Immunoblotting of CD36 and Glut1 and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). I - L Representative Immunoblotting of CD73, TNAP, and PAP and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). M = male; MH = male + HH; F = female; FH = female + HH; MI = male + APCP; FI = female + APCP; MHI = male + HH + APCP; FHI = female + HH + APCP. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Membranes were blocked in TBST with 2% BSA, and proteins of interest were blotted with the antibodies listed here: Glut1 (E4S6I) (Rabbit mAb #73015; CD36 (NB400-144) Novus Biologicals; 1:2000); Cleavage Caspase 3 (Asp175) (D3E9); Cell Signaling Technology; #9579 Rabit mAb;1:1000), CD73 (Abcam; Ab175396; 1:1000), anti-GATA4 (Abcam; ab84593; 1:1000), TNAP (ALPL) Rabbit mAb (A0514); PAP (PSAP) Rabbit mAb (A21195); GAPDH (Proteintech; 10494–1-AP; 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech; SA00001-2; 1:1000).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner

doi: 10.1186/s12964-024-01535-8

Figure Lengend Snippet: Double inhibition of PAP and CD73 in male mice, mitigated glycolytic shift and proinflammatory response. A - C Representative Immunoblotting of CD73, TNAP, and PAP and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). E ELISA analysis of Extracellular Adenosine level (n = 4 mice/group). F - H Representative Oil Red O (ORO) and Periodic Acid Schiff (PAS) staining of myocardial sections and their respective graphical presentations showing lipid and glycogen deposition percentages ( n = 4–6 sections per 4–5 mice per group) Scale bar, 50 μm. I - K Representative Immunoblotting of CD36 and Glut1 and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). L - N Representative immunohistochemical staining and graphical presentation of macrophage CD86 and CD206-positive cells assessed from the myocardial sections ( n = 4–5 hearts per group) Scale bar, 50 μm. O - Q Inflammatory cytokines; Interleukin TNF-α, IL-10, and transforming growth factor (TGF)-β concentrations assessed by ELISA using myocardia lysates. All ELISA were performed in triplicates ( n = 4–5 mice per group). M = male; MH = male + HH; F = female; FH = female + HH; MI = male + APCP; FI = female + APCP; MHI = male + HH + APCP; FHI = female + HH + APCP. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Membranes were blocked in TBST with 2% BSA, and proteins of interest were blotted with the antibodies listed here: Glut1 (E4S6I) (Rabbit mAb #73015; CD36 (NB400-144) Novus Biologicals; 1:2000); Cleavage Caspase 3 (Asp175) (D3E9); Cell Signaling Technology; #9579 Rabit mAb;1:1000), CD73 (Abcam; Ab175396; 1:1000), anti-GATA4 (Abcam; ab84593; 1:1000), TNAP (ALPL) Rabbit mAb (A0514); PAP (PSAP) Rabbit mAb (A21195); GAPDH (Proteintech; 10494–1-AP; 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech; SA00001-2; 1:1000).

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: The Kidney Contains Ontogenetically Distinct Dendritic Cell and Macrophage Subtypes throughout Development That Differ in Their Inflammatory Properties

doi: 10.1681/ASN.2019040419

Figure Lengend Snippet: The adult kidney contains four phenotypically distinct subsets of MPs with Clec9a-expression history. (A–C) Kidney leukocytes from 10- to 12-week-old Clec9acre/creRosaYFP mice were analyzed by flow cytometry. (A) Live CD45.2+ MHCII+ cells were gated as indicated and subdivided into CD11c+CD64− and CD64+ cells. CD11c+CD64− cells were further analyzed for XCR-1 and CD11b expression to identify cDC1s and cDC2s, respectively. CD64+ cells were further divided into F4/80hi and CD11bhi cells. (B) The percentage of YFP+ cells in the indicated populations (see also Supplemental Figure 1A) is shown. (C) Representative t-distributed stochastic neighbor embedding (tSNE) of kidney leukocytes. Cells were clustered independently of their YFP labeling and manually gated populations were overlaid on the tSNE plot in the indicated colors. Blue-to-red gradient indicates increasing intensity of marker expression. (D) Left panel shows the percentage of tomato+ cells in each population; right panel, CX3CR-1-GFP expression in cDC1s, cDC2s, as well as F4/80hi and CD11bhi cells, further divided into tomato+ and tomato− cells. Gray traces represent GFP fluorescence in control mice lacking the CX3CR-1-GFP allele. (E) Renal YFP+ and YFP− F4/80hi cells were analyzed for MERTK and TIM-4 expression by flow cytometry. Expression of these markers on splenic RPMs is shown as positive control. (F) Renal cDC1s, cDC2s, YFP+ and YFP− F4/80hi cells, as well as YFP+ and YFP− CD11bhi cells from Clec9acre/creRosaYFP mice were analyzed for IRF4, IRF8, ZBTB46, and F4/80 expression. Gray traces represent staining with isotype-matched control antibodies. (B–D) Each dot represents one mouse, horizontal bars represent mean, error bars represent SD. ***P<0.001, ****P<0.0001.

Article Snippet: Anti-CD64 (50086-R027, rabbit IgG) was purchased from Sino Biologic, cleaved caspase-3 (clone D3E9, rabbit IgG) was purchased from Cell Signaling Technologies, goat anti-rabbit IgG-AF555 (A21429) was purchased from Thermo Fisher, and goat anti-rabbit IgG-AF488 (111-545-144) secondary antibody was ordered from Jackson ImmunoResearch.

Techniques: Expressing, Flow Cytometry, Labeling, Marker, Fluorescence, Positive Control, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: The Kidney Contains Ontogenetically Distinct Dendritic Cell and Macrophage Subtypes throughout Development That Differ in Their Inflammatory Properties

doi: 10.1681/ASN.2019040419

Figure Lengend Snippet: Renal MP populations exhibit dynamic age-dependent changes. (A–C) Kidneys isolated from Clec9acre/creRosaYFP mice on PND2, PND14, PND28, and 10–12 weeks after birth were analyzed by flow cytometry. (A) Cells were first gated on live CD45.2+ cells and further subdivided into MHCIIneg and MHCII+ cells (top row). MHCII+ cells were further analyzed for CD11c and CD64 expression (second row). MHCII+CD64+ cells (third row) and MHCIIneg cells (bottom row) were further analyzed for CD11b and F4/80 expression. (B) Frequency and total number of kidney CD64− cDCs, CD11bhi, F4/80hi and MHCIInegF4/80hi cells at the indicated ages are shown. (C) Percentage of YFP+ cells in each population from Clec9acre/creRosaYFP mice at the indicated ages. Each dot represents one mouse, horizontal lines indicate mean, error bars represent SD. ***P<0.001, ****P<0.0001, only statistically significant differences are marked.

Article Snippet: Anti-CD64 (50086-R027, rabbit IgG) was purchased from Sino Biologic, cleaved caspase-3 (clone D3E9, rabbit IgG) was purchased from Cell Signaling Technologies, goat anti-rabbit IgG-AF555 (A21429) was purchased from Thermo Fisher, and goat anti-rabbit IgG-AF488 (111-545-144) secondary antibody was ordered from Jackson ImmunoResearch.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: The Kidney Contains Ontogenetically Distinct Dendritic Cell and Macrophage Subtypes throughout Development That Differ in Their Inflammatory Properties

doi: 10.1681/ASN.2019040419

Figure Lengend Snippet: MHCII+ cells in the kidney are Myb dependent and do not arise from embryonic progenitors. (A) Kidneys from Myb−/− and wild-type littermate control mice were analyzed by flow cytometry on E16.5. Left: CD11c and MHCII expression of live CD45.2+ cells in mouse embryos of the indicated genotype is shown. Right: The frequency of MHCII+ and MHCIInegF4/80hi cells among live CD45.2+ cells is shown. (B) CD45.2+Mx-1creMybflox/flox were treated with serial polyI:C injections to induce deletion of HSCs and subsequently transplanted with bone marrow from congenic CD45.1+ wild-type mice. The percentage of donor-derived CD45.1+ cells in the indicated populations in the liver and kidney is shown. (C) Csf1rMer-iCre-Mer females were mated with male Rosa+/YFP mice and injected with 4-hydroxytamoxifen (OH-TAM) on E8.5. On E18.5 and 2 weeks after birth, kidneys and liver from offspring mice were analyzed for YFP expression by flow cytometry. Renal populations were identified as in Figure 4A. Liver Kupffer cells were identified as live CD45.2+F4/80hi cells. The percentage of YFP-positive cells in the indicated populations in kidneys and liver is shown. (D) Renal MHCII+F4/80hi and MHCIInegF4/80hi cells from 2-week-old mice were analyzed for MERTK and TIM-4 expression by flow cytometry. Gray traces represent staining with isotype-matched control antibodies. (E) Renal leukocytes from 2-week-old mice were stimulated in vitro with R848, LPS, or zymosan (Zym) for 6 hours and analyzed for intracellular TNF production by flow cytometry. The frequency of TNF-positive MHCII+F4/80hi and MHCIInegF4/80hi cells was calculated and plotted. (F–H) Kidney sections from two-week-old Clec9acreRosaTom mice were analyzed by histo-cytometry. (F) Immunofluorescence image of the following markers: F4/80 (red), MHCII (green), CD11b (magenta), and CD64 (blue). Scale bar, 100 µm. (G) Histo-cytometry was used to identify the x and y position of MHCIInegF4/80hi and MHCII+F4/80hi cells in the kidney sections. Lines separating renal medulla and cortex were drawn by hand based on tissue structure, autofluorescence properties, and presence of glomeruli. (H) The frequency of MHCIIneg F4/80hi and MHCII+F4/80hi cells located in the renal cortex and medulla was quantified using gates on the renal cortex or medulla in histo-cytometry analysis and plotted. Each dot represents one mouse. Horizontal bars represent mean, error bars represent SD. ****P<0.0001.

Article Snippet: Anti-CD64 (50086-R027, rabbit IgG) was purchased from Sino Biologic, cleaved caspase-3 (clone D3E9, rabbit IgG) was purchased from Cell Signaling Technologies, goat anti-rabbit IgG-AF555 (A21429) was purchased from Thermo Fisher, and goat anti-rabbit IgG-AF488 (111-545-144) secondary antibody was ordered from Jackson ImmunoResearch.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Injection, Staining, In Vitro, Cytometry, Immunofluorescence

Journal: Journal of the American Society of Nephrology : JASN

Article Title: The Kidney Contains Ontogenetically Distinct Dendritic Cell and Macrophage Subtypes throughout Development That Differ in Their Inflammatory Properties

doi: 10.1681/ASN.2019040419

Figure Lengend Snippet: Renal MHCIInegF4/80hi cells appear in adult kidneys after cisplatin and ischemia-induced AKI due to a phenotypic switch of MHCII+F4/80hi cells. (A–D) Clec9acre/creRosaYFP mice, 10 weeks of age, were injected i.p. with 15 mg/kg cisplatin and analyzed 72 hours later. (A) Serum creatinine and BUN levels are shown. (B–D) Kidney leukocytes from cisplatin-treated Clec9acre/creRosaYFP mice were analyzed by flow cytometry. (B) Representative tSNE analysis of kidney leukocytes 3 days after cisplatin treatment. Left panel: Cells were clustered independently of YFP labeling and manually gated populations were overlaid on the tSNE plot in the indicated colors. Right panel: The intensity of YFP expression in the indicated populations. Blue-to-red gradient indicates level of marker expression. (C and D) CD45.2+ cells were subdivided into MHCIIneg and MHCII+ cells. MHCII+ cells were further analyzed for CD11c and CD64 expression. After exclusion of Ly6Cneg cells, MHCII+CD64+ cells (first row) and MHCIIneg cells (second row) were further analyzed for CD11b and F4/80 expression. (D) The percentage of YFP+ labeling in the indicated renal leukocyte populations from cisplatin-treated mice is shown. (E) Kidney sections from cisplatin-treated mice were analyzed by immunofluorescence microscopy for the following markers: CD11b (magenta), F4/80 (red), MHCII (green), and cleaved caspase-3 (cyan). Representative cutouts of renal cortex and medulla are shown. Scale bar, 100 µm. (F) Quantification of MHCII+F4/80hi and MHCIInegF4/80hi cells in the renal cortex and medulla in cisplatin-treated mice. Each dot represents the average amount of cells per field from one biologic replicate. (G and H) FACS analysis of renal leukocytes from Clec9acre/creRosaYFP mouse 72 hours after unilateral ischemia-reperfusion injury. (G) Representative tSNE analyses of kidney leukocytes from ischemic and nonischemic control kidneys. Left panel: Cells were clustered independently of YFP labeling and manually gated populations were overlaid on the tSNE plot in the indicated colors. Right panel: YFP expression in the indicated populations. Blue-to-red gradient indicates increasing intensity of marker expression. (H) The percentage of YFP+ cells in renal MHCII+F4/80hi cells from nonischemic control kidneys and MHCIIneg and MHCII+F4/80hi cells from ischemic kidneys is shown. Each dot represents one mouse. Horizontal bars represent mean, error bars represent SD. ***P<0.001. IRI, ischemic-reperfusion injury; NaCl, sodium chloride.

Article Snippet: Anti-CD64 (50086-R027, rabbit IgG) was purchased from Sino Biologic, cleaved caspase-3 (clone D3E9, rabbit IgG) was purchased from Cell Signaling Technologies, goat anti-rabbit IgG-AF555 (A21429) was purchased from Thermo Fisher, and goat anti-rabbit IgG-AF488 (111-545-144) secondary antibody was ordered from Jackson ImmunoResearch.

Techniques: Injection, Flow Cytometry, Labeling, Expressing, Marker, Immunofluorescence, Microscopy

Journal: Developmental neurobiology

Article Title: Temporally Restricted Death and the Role of p75NTR as a Survival Receptor in the Developing Sensory Nervous System

doi: 10.1002/dneu.22591

Figure Lengend Snippet: (A-B) Representative immunofluorescence against Ki67 in E14.5 DRG with quantification of Ki67+ cells in DRGs from WT or p75NTR−/− . *** denotes p<0.001 analyzed through two-way Anova with Sidak post hoc and no significance (p>0.05 - n.s.) was found between the number of ki67+ cells in WT and p75NTR-/- within any timepoint.

Article Snippet: Primary antibodies used in this study: Goat Anti-TrkA (R&D, AF1056, 1:200 or 0.2ug/mL, RRID:AB_2283049), Goat Anti-TrkB (R&D, AF1494, 1:100 or 0.2ug/mL, RRID:AB_2155264), Goat Anti-TrkC (R&D, AF1404, 1:500, RRID:AB_2155412), Rabbit Anti-Cleaved Caspase-3 (Cell Signaling, 9661S, 1:200, RRID:AB_2341188), Mouse Anti-Islet1/2 (DSHB, 39.4D5, 1:100, RRID:AB_528173), Goat Anti-Ret (Neuromics, GT15002, 1:1000, RRID:AB_1622006), Rabbit Anti-Ki67 (Abcam, AB15580, 1:300, RRID:AB_805388), Rabbit Anti-Sox10 (Gift from S. Kucenas, 1:5,000), Rabbit Anti-p75NTR (Promega, G3231, 1:250, RRID:AB_430853), Rabbit Anti-BFABP (Gift from C. Birchmeier and T. Müller, 1:10,000, Kurtz et al., 1994 ), Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (Alexa Fluor® 488 Conjugate, 9603, 1:50, RRID:AB_11179205).

Techniques: Immunofluorescence

Journal: Developmental neurobiology

Article Title: Temporally Restricted Death and the Role of p75NTR as a Survival Receptor in the Developing Sensory Nervous System

doi: 10.1002/dneu.22591

Figure Lengend Snippet: (A) Representative co-immunofluorescence of E11.5 lower lumbar DRGs with anti-neurotrophin receptors (NTR) (TrkA/B/C/or Ret) or anti-Ret and sensory neuron marker anti-Islet1/2. Scale bar, 20μm.

Article Snippet: Primary antibodies used in this study: Goat Anti-TrkA (R&D, AF1056, 1:200 or 0.2ug/mL, RRID:AB_2283049), Goat Anti-TrkB (R&D, AF1494, 1:100 or 0.2ug/mL, RRID:AB_2155264), Goat Anti-TrkC (R&D, AF1404, 1:500, RRID:AB_2155412), Rabbit Anti-Cleaved Caspase-3 (Cell Signaling, 9661S, 1:200, RRID:AB_2341188), Mouse Anti-Islet1/2 (DSHB, 39.4D5, 1:100, RRID:AB_528173), Goat Anti-Ret (Neuromics, GT15002, 1:1000, RRID:AB_1622006), Rabbit Anti-Ki67 (Abcam, AB15580, 1:300, RRID:AB_805388), Rabbit Anti-Sox10 (Gift from S. Kucenas, 1:5,000), Rabbit Anti-p75NTR (Promega, G3231, 1:250, RRID:AB_430853), Rabbit Anti-BFABP (Gift from C. Birchmeier and T. Müller, 1:10,000, Kurtz et al., 1994 ), Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (Alexa Fluor® 488 Conjugate, 9603, 1:50, RRID:AB_11179205).

Techniques: Immunofluorescence, Marker