Journal: Biomolecules

Article Title: HIIT Promotes M2 Macrophage Polarization and Sympathetic Nerve Density to Induce Adipose Tissue Browning in T2DM Mice

doi: 10.3390/biom14030246

Figure Lengend Snippet: Primers used for quantitative real-time PCR analysis.

Article Snippet: The sections were incubated in 5% bovine serum albumin for 2 h, followed by overnight incubation at 4 °C with the following primary antibodies: rat anti-Mac2 antibody (1:500, Cedarlane, Burlington, ON, Canada, CL8942AP), rabbit anti-iNOS antibody (1:500, Abcam, Cambridge, UK, ab15323), rabbit anti-Arg1 antibody (1:500, Cell Signaling Technology, Boston, MA, USA, 93668), and sheep anti-TH antibody (1:500, Novus Biologicals, CO, USA, NB300-110).

Techniques: Real-time Polymerase Chain Reaction

Journal: Biomolecules

Article Title: HIIT Promotes M2 Macrophage Polarization and Sympathetic Nerve Density to Induce Adipose Tissue Browning in T2DM Mice

doi: 10.3390/biom14030246

Figure Lengend Snippet: The effects of exercise intervention on macrophage infiltration and polarization in T2DM mice. ( A ) Representative images of Mac2 immunofluorescence staining of iWAT in control, T2DM, and T2DM-EX mice, scale bar = 50 μm. ( B ) Percentage of Mac2-positive area in total image, 3 samples per group, 6 fields per section. ( C – E ) The mRNA expression levels of pan-macrophage markers (Mac2, F4/80, and CD11b). ( F ) Representative images showing the double-positive staining of Mac2 and iNOS, Mac2 and Arg1. Scale bar = 50 μm. White arrows indicate Mac2 single-positive cells; red arrows indicate the double-positive cells. Green: Mac2; red: iNOS or Arg1; blue: nucleus stained by DAPI. ( G ) The ratio of double-positive cells to Mac2 single-positive cells. M1 represents Mac2 + iNOS + and M2 represents Mac2 + Arg1 + . Three samples per group, 6 fields per section. ( H ) The mRNA levels of M1-type macrophage markers (iNOS, CD11c) in iWAT. ( I ) The mRNA levels of M2-type macrophage markers (CD206, Arg1) in iWAT. * p < 0.05, ** p < 0.01. Data are expressed as mean ± SEM.

Article Snippet: The sections were incubated in 5% bovine serum albumin for 2 h, followed by overnight incubation at 4 °C with the following primary antibodies: rat anti-Mac2 antibody (1:500, Cedarlane, Burlington, ON, Canada, CL8942AP), rabbit anti-iNOS antibody (1:500, Abcam, Cambridge, UK, ab15323), rabbit anti-Arg1 antibody (1:500, Cell Signaling Technology, Boston, MA, USA, 93668), and sheep anti-TH antibody (1:500, Novus Biologicals, CO, USA, NB300-110).

Techniques: Immunofluorescence, Staining, Control, Expressing

Journal: Biomolecules

Article Title: HIIT Promotes M2 Macrophage Polarization and Sympathetic Nerve Density to Induce Adipose Tissue Browning in T2DM Mice

doi: 10.3390/biom14030246

Figure Lengend Snippet: Exercise-induced aggregation of M2-like macrophages surrounding sympathetic nerves. Immunofluorescence staining of iWAT for TH (white), Mac2 (green), and iNOS (red, A ) or Arg1 (red, B ), and stained nuclei with DAPI (blue). Scale bar = 50 μm. ( C – E ) In pan-macrophages (Mac2 + ), the proportion of TH + Mac2 + iNOS + cells or TH + Mac2 + Arg1 + cells and the ratio of the two. Three samples per group, 6 fields per section. * p < 0.05, ** p < 0.01. Data are expressed as mean ± SEM.

Article Snippet: The sections were incubated in 5% bovine serum albumin for 2 h, followed by overnight incubation at 4 °C with the following primary antibodies: rat anti-Mac2 antibody (1:500, Cedarlane, Burlington, ON, Canada, CL8942AP), rabbit anti-iNOS antibody (1:500, Abcam, Cambridge, UK, ab15323), rabbit anti-Arg1 antibody (1:500, Cell Signaling Technology, Boston, MA, USA, 93668), and sheep anti-TH antibody (1:500, Novus Biologicals, CO, USA, NB300-110).

Techniques: Immunofluorescence, Staining

Journal: Cell and tissue research

Article Title: Prolonged high fat diet ingestion, obesity, and type 2 diabetes symptoms correlate with phenotypic plasticity in myenteric neurons and nerve damage in the mouse duodenum

doi: 10.1007/s00441-015-2132-9

Figure Lengend Snippet: Antibody manufacturer and dilutions used for immunohistochemistry

Article Snippet: All dilution information for primary antibodies, secondary antibodies and dyes can be found in . table ft1 table-wrap mode="anchored" t5 caption a7 Primary Antibody, Dilution Secondary Antibody, Dilution Dye, Dilution All Neurons Ms monoclonal HuC/HuD, 1:200 Molecular Probes, Eugene, OR {"type":"entrez-nucleotide","attrs":{"text":"A21271","term_id":"514139","term_text":"A21271"}} A21271 Dk anti Ms IgG Dylight 488, 1:300 Jackson mmunoResearch, West Grove, PA 715-485-150 N/A Inhibitory Motor Neurons Rb polyclonal VIP, 1:1000 Abcam, Cambridge, MA Ab43841 Biotinylated Gt anti Rb IgG, 1:300 Vector Labs, Burlingame, CA BA1000 Streptavidin Alexa Fluor 488 conjugate, 1:600; Jackson ImmunoResearch, West Grove, PA 016-540-084 Rb polyclonal nNOS, 1:500 Invitrogen, Camarillo, CA #61-7000 Biotinylated Gt anti Rb IgG, 1:300 Vector Labs, Burlingame, CA BA1000 Streptavidin Cy3 conjugate, 1:600 Life Technologies, Grand Island, NY SA1010 Sheep polyclonal nNOS, 1:500 Abcam, Cambridge, MA Ab6175 Dk anti Sheep IgG Dylight 594, 1:300 Jackson ImmunoResearch, West Grove, PA 715-485-150 N/A Excitatory Motor Neurons Gt polyclonal ChAT, 1:200 Millipore, Billerica, MA AB144P Biotinylated Rb anti Gt IgG, 1:300 Vector Labs, Burlingame, CA BA5000 N/A Rt monoclonal Substance P, 1:150 Millipore, Billerica, MA MAB356 Biotinylated Gt anti Rt, 1:300 Invitrogen, Camarillo, CA #62–9540 Streptavidin Cy3 conjugate, 1:600 Life Technologies, Grand Island, NY SA1010 Sensory Neurons Rabbit polyclonal anti-Calbindin D-28k, 1:1250 CH-1723, Marly 1 (Switzerland) CB 38 Biotinylated Gt anti Rb IgG, 1:300 Vector Labs, Burlingame, CA BA1000 Streptavidin Cy3 conjugate, 1:600 Life Technologies, Grand Island, NY SA1010 Rb polyclonal CGRP, 1:200 Sigma Aldrich, St. Louis, MO # c8198 Biotinylated Gt anti Rb IgG, 1:300 Vector Labs, Burlingame, CA BA1000 Streptavidin Cy3 conjugate, 1:600 Life Technologies, Grand Island, NY SA1010 Apoptosis Rb polyclonal anti-cleaved Caspase-3, Cell Signaling Technology (ASP 175) Dk anti rabbit IgG Dylight 488, 1:300 N/A Open in a separate window Ms: Mouse, Dk: Donkey, Rb: Rabbit, Gt: Goat, Rt: Rat Antibody manufacturer and dilutions used for immunohistochemistry To ensure the specificity of staining, co-localization of nucleated neuronal cells was confirmed through DAPI counterstaining, and primary and secondary antibody negative control staining was performed for all antibodies used.

Techniques: Plasmid Preparation

Journal: Nature Neuroscience

Article Title: Distinct origin and region-dependent contribution of stromal fibroblasts to fibrosis following traumatic injury in mice

doi: 10.1038/s41593-024-01678-4

Figure Lengend Snippet: a , Schematic outline and experimental timeline used for lineage tracing of GLAST- and Col1a1 -expressing perivascular cells following LPS injection into the uninjured spinal cord gray matter and white matter. b , c , Stereotactic injection of LPS into the spinal cord gray matter of GLAST-CreER T2 ; R26R-tdTomato ; Pdgfrb-eGFP ( b ) and Col1a1 - CreER T2 ; R26R-tdTomato ; Pdgfrb - eGFP ( c ) mice triggers the recruitment of MAC2 + macrophages/microglia and local accumulation of tdTomato + EGFP + stromal fibroblasts at 5 days after injection. b′ , Close-up view of the boxed region in b showing that most Pdgfrb -EGFP + stromal fibroblasts are tdTomato + (yellow arrowheads), with only a few tdTomato − EGFP + cells (white arrowhead) located off blood vessels (podocalyxin + ). c′ , Magnified image of the boxed region in c showing that almost all Pdgfrb -EGFP + stromal fibroblasts are tdTomato − (white arrowheads), indicating that Col1a1 -expressing perivascular fibroblasts (tdTomato + EGFP + cells; yellow arrowheads) do not contribute substantially to gray matter scarring. d , e , Focal injection of LPS into the spinal cord ventral white matter of GLAST-CreER T2 ; R26R-tdTomato ; Pdgfrb-eGFP ( d ) and Col1a1 - CreER T2 ; R26R-tdTomato ; Pdgfrb-eGFP ( e ) mice leads to recruitment of MAC2 + macrophages/microglia and local accumulation of tdTomato + EGFP + stromal fibroblasts at 5 days after injection. d′ , e′ , Magnified images of the boxed regions in d and e showing several tdTomato + EGFP + stromal fibroblasts (yellow arrowheads) and tdTomato − EGFP + stromal cells (white arrowheads) in GLAST-CreER T2 ; R26R-tdTomato ; Pdgfrb-eGFP ( d′ ) and Col1a1-CreER T2 ; R26R-tdTomato ; Pdgfrb-eEGFP ( e′ ) mice. f , g , Quantification of the inflammatory response by MAC2 + cell area ( f ) and percentage of tdTomato + PDGFRβ + cells out of the total PDGFRβ + stromal cells ( g ) at 5 days after intraspinal injection of LPS into the gray matter or white matter of GLAST-CreER T2 ; R26R-tdTomato ; Pdgfrb-eGFP ( GLAST-CreER T2 ) and Col1a1-CreER T2 ; R26R-tdTomato ; Pdgfrb-eGFP ( Col1a1-CreER T2 ) mice. ( b′ , c′ , d′ , e′ ) Upper panel: tdTom, EGFP and Pdx labelling; lower panel: tdTom and Pdx labelling. Scale bars: 500 µm ( b , c , d , e ) and 100 µm ( b′ , c′ , d′ , e′ ). Data are shown as mean ± s.d. Col1a1-CreER T2 : n = 3 (gray matter), n = 4 (white matter); GLAST-CreER T2 : n = 4 (gray matter), n = 3 (white matter) animals in f . C ol1a1-CreER T2 : n = 3 (gray matter), n = 4 (white matter); G LAST-CreER T2 : n = 7 (gray matter), n = 6 (white matter) animals in g . NS (gray matter) = 0.5719, NS (white matter) = 0.9788 ( f ) and NS = 0.1624, *** P = 0.007, **** P < 0.0001 ( g ) by one-way ANOVA followed by Tukey’s multiple-comparisons test. Dashed lines in b – e outline the spinal cord gray matter. Images are representative of two independent experiments. All images show transverse sections. Source data are provided as a source data file.

Article Snippet: The primary antibodies used were GFP (1:2,000, chicken, Aves Labs, GFP-1020; 1:2,000, sheep, Bio-Rad, 4745-1051), RFP (red fluorescent protein; 1:250, chicken, Novus Biologicals, NBP1-97371), PDGFRβ (1:200, rabbit, Abcam, ab32570; 1:100, rat, eBioscience, 14-1402-82), podocalyxin (1:200, goat, R&D Systems, AF1556), αSMA (1:200, rabbit, Abcam, ab5694), SM22α (also known as transgelin; 1:500, rabbit, Abcam, ab14106), Ki67 (1:2,000, rat, eBioscience, 14-5698), NG2 chondroitin sulfate proteoglycan (1:200, rabbit, Millipore, AB5320; no Triton X-100) and MAC2 (also known as galectin-3; 1:500, rat directly conjugated to biotin, Cedarlane Labs, CL8942B).

Techniques: Expressing, Injection

Journal: Frontiers in Immunology

Article Title: Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep

doi: 10.3389/fimmu.2023.1243204

Figure Lengend Snippet: CD4 + and CD8 + T cells in PBMCs. Peripheral blood samples were collected at different immunization times, and CD4 + and CD8 + T cells in PBMCs were analyzed using flow cytometry. (A) Proportion of CD4 + and CD8 + T cells in PBMCs of each group at week 8. (B, C) show the trend of the proportion of CD4 + and CD8 + T cells at different times, respectively. (D) The ratio of CD4 + to CD8 + T cells in each group of PBMCs at week 8. (E) The trend of the ratio of CD4 + to CD8 + T cells at different times. Data were obtained from 9 sheep, and results are presented as mean ± SD ( ns , P > 0.05).

Article Snippet: To determine the cellular phenotypes of PBMCs, cells were directly stained with mouse anti-sheep CD4 (Clone: 44.38) and mouse anti-sheep CD8 (Clone: 38.65) antibodies (Bio-Rad AbD Serotec, Munich, USA), and incubated at 4°C for 30 min in the dark, followed by two washes with washing buffer.

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep

doi: 10.3389/fimmu.2023.1243204

Figure Lengend Snippet: Cytokines produced in PBMCs by flow cytometry. PBMCs were stimulated with rEg.P29, and the cells were collected and labeled with antibodies to detect IFN-γ and IL-17A production by CD4 + and CD8 + T cells using flow cytometry. (A) Representative flow scatter plots for detecting IFN-γ production by CD4 + and CD8 + T cells. (B, D) show the IFN-γ production by CD4 + and CD8 + T cells in each group at week 8, respectively. (C, E) show the tendency of IFN-γ production by CD4 + and CD8 + T cells at different times, respectively. (F) Representative flow scatter plots for detecting IL-17A production by CD4 + and CD8 + T cells. (G, I) show the IL-17A production by CD4 + and CD8 + T cells in each group of samples at week 8, respectively. (H, J) show the tendency of IL-17A production by CD4 + and CD8 + T cells at different times, respectively. Data were obtained from 9 sheep, and results are presented as mean ± SD (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: To determine the cellular phenotypes of PBMCs, cells were directly stained with mouse anti-sheep CD4 (Clone: 44.38) and mouse anti-sheep CD8 (Clone: 38.65) antibodies (Bio-Rad AbD Serotec, Munich, USA), and incubated at 4°C for 30 min in the dark, followed by two washes with washing buffer.

Techniques: Produced, Flow Cytometry, Labeling

Journal: Frontiers in Immunology

Article Title: Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep

doi: 10.3389/fimmu.2023.1243204

Figure Lengend Snippet: Cytokines produced in spleen lymphocytes by flow cytometry. Spleen lymphocytes were obtained after euthanasia of sheep, cells were labeled with antibodies after rEg.P29 stimulation, and IFN-γ and IL-17A production by CD4 + and CD8 + T cells was detected using flow cytometry. (A) Representative flow scatter plots for detecting IFN-γ production by CD4 + and CD8 + T cells. (B, C) show the IFN-γ production by CD4 + and CD8 + T cells in each group, respectively. (D) Representative flow scatter plots for detecting IL-17A production by CD4 + and CD8 + T cells. (E, F) show the IL-17A production by CD4 + and CD8 + T cells in each group, respectively. Data were obtained from 7 sheep, and results are presented as mean ± SD (**** P < 0.0001).

Article Snippet: To determine the cellular phenotypes of PBMCs, cells were directly stained with mouse anti-sheep CD4 (Clone: 44.38) and mouse anti-sheep CD8 (Clone: 38.65) antibodies (Bio-Rad AbD Serotec, Munich, USA), and incubated at 4°C for 30 min in the dark, followed by two washes with washing buffer.

Techniques: Produced, Flow Cytometry, Labeling

Journal: Frontiers in Immunology

Article Title: Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep

doi: 10.3389/fimmu.2023.1243204

Figure Lengend Snippet: Cytokines produced in lymphocytes of mesenteric lymph nodes by flow cytometry. Lymphocytes of mesenteric lymph nodes were obtained after euthanasia of sheep, cells were labeled with antibodies after rEg.P29 stimulation, and IFN-γ, IL-4, and IL-17A production by CD4 + and CD8 + T cells was detected using flow cytometry. (A) Representative flow scatter plots for detecting IFN-γ production by CD4 + and CD8 + T cells. (B, C) show the IFN-γ production by CD4 + and CD8 + T cells in each group, respectively. (D) Representative flow scatter plots for detecting IL-17A production by CD4 + and CD8 + T cells. (E, F) show the IL-17A production by CD4 + and CD8 + T cells in each group, respectively. Data were obtained from 7 sheep, and results are presented as mean ± SD (**** P < 0.0001).

Article Snippet: To determine the cellular phenotypes of PBMCs, cells were directly stained with mouse anti-sheep CD4 (Clone: 44.38) and mouse anti-sheep CD8 (Clone: 38.65) antibodies (Bio-Rad AbD Serotec, Munich, USA), and incubated at 4°C for 30 min in the dark, followed by two washes with washing buffer.

Techniques: Produced, Flow Cytometry, Labeling

Journal: Frontiers in Immunology

Article Title: Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep

doi: 10.3389/fimmu.2023.1243204

Figure Lengend Snippet: Proliferation of CD4 + and CD8 + T cells. PBMCs were labeled with CFSE, stimulated in vitro with rEg.P29, and the decrease in CFSE fluorescence intensity of labeled cells was detected using flow cytometry to assess the proliferation of CD4 + and CD8 + T cells. (A) Histogram plots of CFSE fluorescence of lymphocytes, CD4 + , and CD8 + T cells. (B–D) represent the proliferation frequencies of lymphocytes, CD4 + , and CD8 + T cells, respectively. Data were obtained from 5 sheep, and results are presented as mean ± SD (**** P < 0.0001).

Article Snippet: To determine the cellular phenotypes of PBMCs, cells were directly stained with mouse anti-sheep CD4 (Clone: 44.38) and mouse anti-sheep CD8 (Clone: 38.65) antibodies (Bio-Rad AbD Serotec, Munich, USA), and incubated at 4°C for 30 min in the dark, followed by two washes with washing buffer.

Techniques: Labeling, In Vitro, Fluorescence, Flow Cytometry