Journal: Cell Reports Medicine

Article Title: Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis

doi: 10.1016/j.xcrm.2020.100056

Figure Lengend Snippet:

Article Snippet: Rabbit anti-αSMA monoclonal antibody (EPR5368) IHC , Abcam , Cat#Ab124964; RRID: AB_11129103.

Techniques: Transduction, Ligation, Recombinant, Injection, Protease Inhibitor, Mass Spectrometry, Staining, Enzyme-linked Immunosorbent Assay, Software, Western Blot

Journal: JCI Insight

Article Title: Mutations in EPHB4 cause human venous valve aplasia

doi: 10.1172/jci.insight.140952

Figure Lengend Snippet: ( A ) Localization of PECAM1 (blue), and either Cx37 or Cx43, as indicated (magenta) around VFCs on P0 in heterozygous Efnb2GFP (green) mice. As expected, on P0 Cx37 was localized to Efnb2GFP-expressing VFCs, primarily forming large gap junction plaques (examples indicated between arrowheads) and Cx43 was localized to endothelium upstream of these VFCs (region to the left of the arrowheads). Smaller plaques are also identifiable. ( B ) Localization of PECAM1 (blue), Prox1 (red), and either Cx37 or Cx43, as indicated (green), after homozygous deletion of Efnb2 . The tightly regulated expression pattern of Cx37 was disrupted, with expression over a wider area (arrowheads) and the typical appearance of larger plaques was lost. The expression pattern of Cx43 was also disrupted and no longer confined to upstream of VFCs (arrowheads; P < 0.05, χ 2 test of the proportion of VVs showing normal [confined] vs. disrupted expression pattern, n ≥ 6 VV per group). ( C and D ) The proportion of proliferating VFCs was assessed by colocalization of Prox1 and Ki67 (arrowheads). Ki67 + VFCs were easily identified in littermate controls, but far fewer proliferating VFCs were identifiable after homozygous Efnb2 deletion. The inferior region of the vein is shown; **** P < 0.00005, unpaired 2-tailed t test, n ≥ 6 VV per group, data are shown as mean ± SEM). Scale bars in A–C : 20 μm. VFCs, valve-forming cells; VVs, venous valves; P0, postnatal day 0.

Article Snippet: The following antibodies were used: rabbit anti-Cx43 (Cell Signaling Technology 3512), Cx37 (CX37A11, Alpha Diagnostics), Prox1 (11-002P, Angiobio), ki67 (ab15580, Abcam), and Golgi apparatus protein 1 (ab103439, Abcam); sheep anti-Foxc2 (AF6989, R&D); goat anti-EphB4 (BAF446, R&D); rat anti-PECAM1 (clone MEC 13.3, BD); and mouse anti-α smooth muscle actin (clone 1A4 conjugated to Cy3, MilliporeSigma).

Techniques: Expressing

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Nav1.7 immunolabeling (IL) of arterioles (Ar), arteriole-venule shunts (AVS) and associated innervation in normal human plantar glabrous skin with Alomone (A, B) or Yale (C) Nav1.7 antibodies (red). Co-labeling of innervation (arrows) as marked with anti-PGP 9.5 (PGP, green, A ) or smooth muscle cells in tunica media (tm) as marked with anti α-smooth muscle actin antibody (αSMA, green, B , C ). Nuclei are DAPI-labeled (blue). Left images (each panel) show only red fluorescence, middle images green; right images show triple-label combinations. Large white rectangles are 2X-enlargements of small rectangles. A-C . Nav1.7-IL is expressed on endothelial cells of tunica intima (red arrowheads) and tm smooth muscle cells as confirmed by double-labeling with anti-αSMA (B, C) . Nav1.7-IL is expressed on virtually all vascular innervation (arrows) in tunica adventitia (ta) as confirmed by anti-PGP 9.5 double-labeling ( A , yellow arrows). N=nerve. D-E . Nav1.7-IL on arteriole endothelial cells shown as 2X-enlargements of areas indicated by white rectangles in B , C . First images (each panel) show Nav1.7-IL on smooth muscle cells in tm and endothelial cells (red arrowheads). The second images show α-SMA co-labeling of only the smooth muscle cells of tm (green). The third images show merge of first and second images with DAPI (blue). Sections re-labeled with anti-PECAM (green) to show co-labeling with Nav1.7 on endothelial cells (yellow arrowheads, fourth and fifth images). F-G . Background Cy3 fluorescence is limited with no primary antibody in arteriole deep in dermis (F) , epidermis (Ep) and upper-dermis (UD) (G) . In F , broken line shows tm perimeter with dotted line around arteriole lumen. In G , broken line indicates basement membrane of epidermis and dotted line indicates boundary of dead and live superficial keratinocyte layers (stratum corneum, sc and stratum granulosum, sg, respectively). Stratum spinosum, ss; stratum basalis, sb; dermal papilla (dp). Scale bars=150μm (A); 100μm (B ,C, F, G) ; 50μm in D , E .

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Immunolabeling, Labeling, Fluorescence

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Digital fluorescence images of Nav1.7 immunolabeling (IL) of arterioles (Ar), arteriole-venule shunts (AVS) and associated innervation in normal human glabrous skin biopsies from the plantar foot (A,C,D) and palmar hand (B) . All sections are labeled with an Alomone (A,C) or Yale (B,D) rabbit anti-rat Nav1.7 antibody revealed by a donkey anti-rabbit Cy3-conjugated secondary antibody (red fluorescence). Secondary antibodies conjugated to Alexa 488 (green fluorescence) were used to assess co-labeling for peptidergic sensory innervation revealed with a sheep anti-human CGRP antibody (A,B) or noradrenergic sympathetic innervation revealed with a sheep anti-human NPY antibody (C,D) . Cell nuclei are labeled with DAPI (blue fluorescence). The left images in each panel show only the red fluorescence, the middle images only the green, and the right images the triple label combinations. Areas outlined in large white rectangles are 2X enlargement of the areas in the small rectangles. A-D . Nav1.7-IL is expressed on the endothelial cells of the tunica intima (red arrowheads) and on smooth muscle cells of the tunica media (tm). A , B . Peptidergic sensory innervation co-expresses Nav1.7-IL and CGRP-IL (yellow straight arrows). Other innervation labeled only with Nav1.7 (red curved arrows) is likely the noradrenergic sympathetic innervation that expresses NPY-IL as shown in C and D . C , D . Noradrenergic sympathetic innervation co-expresses Nav1.7-IL and NPY-IL (yellow curved arrows). Other innervation labeled with only Nav1.7 (red straight arrows) is likely the peptidergic sensory innervation that expresses CGRP-IL as shown in A and B . Scale Bar = 150 μm in A and B , 100 μm in C and D .

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Fluorescence, Immunolabeling, Labeling

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Nav1.7 immunolabeling (IL) of arterioles and associated innervation in normal human palmar glabrous skin biopsies, in alternating sections cut parallel to and through lumen (*) of branched arteriole (A-C) and parallel to arteriole, skimming the interface between tunica media (tm) and tunica adventitia (ta) (D,E) . All sections are labeled with Abcam anti-human Nav1.7 antibody (red). Secondary antibodies conjugated to Alexa 488 (green) were used to assess co-labeling for: smooth muscle cells revealed with mouse anti-α−smooth muscle actin antibody (αSMA, A ); peptidergic sensory innervation revealed with sheep anti-CGRP antibody (yellow straight arrows, B , D ); and noradrenergic sympathetic innervation revealed with sheep anti-NPY antibody (yellow curved arrows, C , E ): Nuclei are labeled with DAPI (blue). Left images in each panel show only the red fluorescence, middle images only green, and right images the triple-label combinations. Areas outlined in large white rectangles (A-C) are 2X enlargements of areas in small rectangles. A-E . A . Nav1.7-IL is expressed on endothelial cells of tunica intima (red arrowheads) and smooth muscle cells of tm as confirmed by double-labeling with anti-αSMA. Nav1.7-IL is expressed on innervation (arrows) in ta, near and at the border with tm. B , D . Peptidergic sensory innervation co-expresses Nav1.7-IL and CGRP-IL (yellow straight arrows). Other innervation labeled only with Nav1.7 (red curved arrows) is likely noradrenergic sympathetic innervation that expresses NPY-IL (C,E) . C , E . Noradrenergic sympathetic innervation co-expresses Nav1.7-IL and NPY-IL (yellow curved arrows). Other innervation labeled with only Nav1.7 (red straight arrows) is likely peptidergic sensory innervation that expresses CGRP-IL as shown in B and D . Scale bar = 100 μm in A-C , 50 μm in D and E .

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Immunolabeling, Labeling, Fluorescence

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Nav1.7 expression in smooth muscle cells of deep dermis arterioles within skin from lateral malleolus of three healthy subjects. Smooth muscle cells (arrowheads) of the arteriole tunica media exhibit robust Nav1.7 (red) immunolabeling (antibody Nav1.7 Y ), which is co-localized with alpha smooth muscle actin (green). Skin samples from 3 healthy subjects (Subject 1: A ; Subject 2: B , C ; Subject 3: D ) display similar patterns of Nav1.7 labeling in the smooth muscle cells of the dermal arterioles. Co-localization of Nav1.7 and alpha smooth muscle actin is yellow in the merged panels. E . Sections incubated without primary antibodies followed by secondary antibodies displayed background levels of immunofluorescence in skin vasculature.

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Expressing, Immunolabeling, Labeling, Incubation, Immunofluorescence

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Digital fluorescence images of Nav1.7 (red) and PGP 9.5 (green) immunolabeling (IL) in the epidermis (Ep) and upper dermis (UD) biopsies of normal human palmar glabrous skin ( A , Abcam anti-Nav1.7) and normal human plantar glabrous skin ( B , Alomone anti-Nav1.7). Stratum corneum, sc; stratum granulosum, sg; stratum spinosum (ss); stratum basalis (sb), dermal papilla (dp). Straight arrows indicate epidermal sensory endings, curved arrows indicate small nerves and individual axons or endings in the upper dermis. The areas enclosed in the large rectangles are 2X enlargements of those in the smaller rectangles. Of all the innervation revealed by anti-PGP 9.5, only some express Nav1.7-IL (yellow straight and curved arrows) whereas other only express PGP 9.5-IL (green straight and curved arrows). Aβ-fiber innervation of a Meissner corpuscle (MC) has little if any Nav1.7-IL. Kertinocytes especially in stratum granulosum label for Nav1.7 (arrowheads) which has a more membranous distribution with the Alomone anti-Nav1.7 antibody, but more diffuse labeling with the Abcam anti-Nav1.7 antibody. Scale bar = 100 μm.

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Fluorescence, Immunolabeling, Labeling

Journal: Physiological Reports

Article Title: The effect of nandrolone decanoate administration on fatigue during a volume‐overload stress in male mice

doi: 10.14814/phy2.70334

Figure Lengend Snippet: Glucocorticoid receptor (GR) expression (a) plantaris and (b) soleus muscles. C, control group; RTS, resistance training sham; RTA, resistance training androgen administration. Representative Western blot images are shown below the quantified data. Original images can be found in Appendix . Individual animal responses and group mean ± SEM are reported.

Article Snippet: Following blocking incubation, the membrane was incubated overnight at 4°C in a cocktail of primary antibodies containing PBS with 5% BSA, rabbit anti‐androgen receptor (AR, Bioss, bs‐0118R) diluted 1:1000, rabbit anti‐tumor necrosis factor‐α (TNFα, Bioss, bs‐0078R) diluted 1:500, mouse anti‐ glucocorticoid receptor (GR, Santa Cruz, sc‐393232) diluted 1:1000, mouse anti‐Glutathione peroxidase (GPX, Santa Cruz, sc‐166,570) diluted 1:1000, and mouse anti‐ α tubulin diluted 1:1000 (Santa Cruz, sc‐5286).

Techniques: Expressing, Muscles, Control, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cellular and Molecular Mechanisms of Kidney Injury in 2,8-Dihydroxyadenine Nephropathy

doi: 10.1681/ASN.2019080827

Figure Lengend Snippet: Primary antibodies for immunohistochemistry or immunofluorescence staining

Article Snippet: Additional histology samples were stained with van Kossa or silver stain, or immunohistochemistry against keratin, CD68, Ki-67, or TNFR1. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Target Protein Antibody Company α -SMA Mouse-anti-human SMA (clone 1A4) Dako/Agilent, M085101–2 (US) Aquaporin-2 Rabbit-anti-AQ2 (polyclonal) Abcam, ab15116 (UK) CD3 Rat-anti-human CD3 (cloneCD3–12) Bio-Rad, MCA1477 (GER) CD11b Rat-anti-mouse CD11b (clone M1/70) PE-labeled eBioscience/Thermo Fisher Scientific, 12–0112–82 (US) CD11c Hamster-anti-mouse CD11c (clone N418) APC-labeled eBioscience/Thermo Fisher Scientific, 17–0114–82 (US) CD13 Rabbit-anti-CD13 (clone EPR4058) Abcam, ab108310 (UK) CD44 Rat-anti-mouse CD44 (clone IM7) BD Pharmingen, 553131 (US) CD68 Mouse-anti-rat CD68 (clone ED1) Bio-Rad, MCA341R (GER) CD68 Mouse-anti-human CD68 (clone KP1) Dako/Agilent, M081401 (US) Collagen III Goat-anti-type III collagen (antiserum) Southern Biotech, 1330–01 (US) Murine monocytes/macrophages (ER-HR3) Rat-anti-mouse monocyte antigen (clone ER-HR3) BMA Biomedicals, T-2012 (CHE) F4/80 Rat-anti-mouse F4/80 (clone CI:A3–1) Bio-Rad, MCA497G (GER) Fibronectin Rabbit-anti-rat fibronectin (polyclonal) Millipore/Merck AB1954 (GER) KIM-1 Goat-anti-mouse TIM-1/KIM-1/HAVCR (polyclonal) R&D Systems, AF1817 (US) Lipocalin-2 (NGAL) Rat-anti-human lipocalin-2/NGAL (clone 220310) R&D Systems, MAB1757 (US) Lipocalin-2 (NGAL) (for double staining) Rabbit-anti-NGAL (polyclonal) Thermo Fisher Scientific, PA5–79590 (US) Cytokeratin (Pan CK) Mouse-anti-pan Cytokeratin (clone Lu5) Abcam, ab17155 (UK) Proliferation (Ki-67, MIB1) Mouse-anti-human (clone KI-67P) Dianova, DIA-505 (GER) Proliferation (PCNA) Mouse-anti-PCNA (clone PC10) Calbiochem/Merck, NA03 (GER) Sm22 α /Transgelin Rabbit-anti-Transgelin (polyclonal) Abcam, ab14106 (UK) Tamm–Horsfall protein Rabbit-anti-human THP (polyclonal) SantaCruz, sc-20631 (GER) TNFR1 Rabbit-anti-TNFR1 (polyclonal) Abcam, ab19139 (UK) TNFR2 Rabbit-anti-TNFR2 (clone EPR1653) Abcam, ab15116 (UK) Vimentin Mouse-anti-vimentin (clone V9) Dako/Agilent, M0725 (US) Open in a separate window α -SMA, α –skeletal muscle actin; AQ-2, aquaporin-2; CD, cluster of differentiation; GER, Germany; CHE, Switzerland; F4/80, EGF-like module–containing mucin-like hormone receptor–like 1; TIM-1, T-cell immunoglobulin and mucin domain 1; HAVCR, Hepatitis A virus cellular receptor; NGAL, neutrophil gelatinase–associated lipocalin, lipocalin-2; CK, cytokeratin; PCNA, proliferating cell nuclear antigen; SM22 α , Smooth muscle protein 22- α /Transgelin; THP, Tamm–Horsfall protein, uromodulin; TNFR, tumor necrosis factor receptor.

Techniques: Immunohistochemistry, Immunofluorescence, Double Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cellular and Molecular Mechanisms of Kidney Injury in 2,8-Dihydroxyadenine Nephropathy

doi: 10.1681/ASN.2019080827

Figure Lengend Snippet: Primary antibodies for immunohistochemistry or immunofluorescence staining

Article Snippet: Additional histology samples were stained with van Kossa or silver stain, or immunohistochemistry against keratin, CD68, Ki-67, or TNFR1. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Target Protein Antibody Company α -SMA Mouse-anti-human SMA (clone 1A4) Dako/Agilent, M085101–2 (US) Aquaporin-2 Rabbit-anti-AQ2 (polyclonal) Abcam, ab15116 (UK) CD3 Rat-anti-human CD3 (cloneCD3–12) Bio-Rad, MCA1477 (GER) CD11b Rat-anti-mouse CD11b (clone M1/70) PE-labeled eBioscience/Thermo Fisher Scientific, 12–0112–82 (US) CD11c Hamster-anti-mouse CD11c (clone N418) APC-labeled eBioscience/Thermo Fisher Scientific, 17–0114–82 (US) CD13 Rabbit-anti-CD13 (clone EPR4058) Abcam, ab108310 (UK) CD44 Rat-anti-mouse CD44 (clone IM7) BD Pharmingen, 553131 (US) CD68 Mouse-anti-rat CD68 (clone ED1) Bio-Rad, MCA341R (GER) CD68 Mouse-anti-human CD68 (clone KP1) Dako/Agilent, M081401 (US) Collagen III Goat-anti-type III collagen (antiserum) Southern Biotech, 1330–01 (US) Murine monocytes/macrophages (ER-HR3) Rat-anti-mouse monocyte antigen (clone ER-HR3) BMA Biomedicals, T-2012 (CHE) F4/80 Rat-anti-mouse F4/80 (clone CI:A3–1) Bio-Rad, MCA497G (GER) Fibronectin Rabbit-anti-rat fibronectin (polyclonal) Millipore/Merck AB1954 (GER) KIM-1 Goat-anti-mouse TIM-1/KIM-1/HAVCR (polyclonal) R&D Systems, AF1817 (US) Lipocalin-2 (NGAL) Rat-anti-human lipocalin-2/NGAL (clone 220310) R&D Systems, MAB1757 (US) Lipocalin-2 (NGAL) (for double staining) Rabbit-anti-NGAL (polyclonal) Thermo Fisher Scientific, PA5–79590 (US) Cytokeratin (Pan CK) Mouse-anti-pan Cytokeratin (clone Lu5) Abcam, ab17155 (UK) Proliferation (Ki-67, MIB1) Mouse-anti-human (clone KI-67P) Dianova, DIA-505 (GER) Proliferation (PCNA) Mouse-anti-PCNA (clone PC10) Calbiochem/Merck, NA03 (GER) Sm22 α /Transgelin Rabbit-anti-Transgelin (polyclonal) Abcam, ab14106 (UK) Tamm–Horsfall protein Rabbit-anti-human THP (polyclonal) SantaCruz, sc-20631 (GER) TNFR1 Rabbit-anti-TNFR1 (polyclonal) Abcam, ab19139 (UK) TNFR2 Rabbit-anti-TNFR2 (clone EPR1653) Abcam, ab15116 (UK) Vimentin Mouse-anti-vimentin (clone V9) Dako/Agilent, M0725 (US) Open in a separate window α -SMA, α –skeletal muscle actin; AQ-2, aquaporin-2; CD, cluster of differentiation; GER, Germany; CHE, Switzerland; F4/80, EGF-like module–containing mucin-like hormone receptor–like 1; TIM-1, T-cell immunoglobulin and mucin domain 1; HAVCR, Hepatitis A virus cellular receptor; NGAL, neutrophil gelatinase–associated lipocalin, lipocalin-2; CK, cytokeratin; PCNA, proliferating cell nuclear antigen; SM22 α , Smooth muscle protein 22- α /Transgelin; THP, Tamm–Horsfall protein, uromodulin; TNFR, tumor necrosis factor receptor.

Techniques: Immunohistochemistry, Immunofluorescence, Double Staining