isolectin ib4 biotin conjugated antibody  (Vector Laboratories)

Journal: bioRxiv

Article Title: Descending locus coeruleus noradrenergic signaling to spinal astrocyte subset is required for stress-induced pain facilitation

doi: 10.1101/2024.11.14.623627

Figure Lengend Snippet: (A and B) PWT before and 30 min after intrathecal administration of NA (0.1 nmol) in wild-type mice pretreated intrathecally with vehicle or CPT (3 nmol) (A: n = 5 mice per group) or in control ( Vgat-Cre mice with intra-SDH of AAV-flex[mCherry]) and SDH-Vgat + IN–A 1 R cKD mice (B: n = 9 mice per group) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; *** P < 0.001). (C and D) PWT before and after acute restraint stress in wild-type mice pretreated intrathecally with vehicle or CPT (C: Vehicle, n = 6 mice; CPT, n = 5 mice) or in control ( Vgat-Cre mice with intra-SDH of AAV-flex[mCherry]) and SDH-Vgat + IN–A 1 R cKD mice (D: Control, n = 6 mice; SDH-Vgat + IN–A 1 R cKD, n = 7 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P <0.0001 vs. vehicle or control group). (E) Representative images of pERK (green) and IB4 (magenta) immunofluorescence in the SDH with or without Aβ fiber stimulation and/or restraint stress. CPT was intrathecally administered 30 min before stress exposure. (F) Quantitative analysis of the number of pERK + cells in superficial laminae of the SDH in each group ( n = 4–5 mice per group; one-way ANOVA with post hoc Tukey’s multiple comparisons test; * P < 0.05, *** P < 0.001). (G) Schematic illustration of the mechanisms of stress-induced pain facilitation highlighting NA signals from LC →SDH -NAergic terminals to Hes5 + astrocytes and Vgat + INs. Data represent mean ± SEM.

Article Snippet: Transverse spinal cord and brain sections (30 μm) were incubated in blocking solution (3% normal goat serum [#S-1000; Vector Laboratories] or normal donkey serum [#017-000-121; Jackson ImmunoResearch]) for 2 hours at room temperature and then incubated for 48 hours at 4°C with primary antibodies: polyclonal rabbit anti-tyrosine hydroxylase (TH; 1:1000; #AB152; Millipore); polyclonal sheep anti-TH (1:1000; #AB1542; Millipore); monoclonal mouse anti-noradrenaline transporter (NET; 1:2000; #NET05-2; Mab Technologies); polyclonal rabbit anti-green fluorescent protein (GFP; 1:1000; #598; MBL International); monoclonal rabbit anti-hemagglutinin (HA)-tag (1:1000; #3724; Cell Signaling); monoclonal rat anti-glial fibrillary acidic protein (GFAP; 1:2000; #13-0300; Invitrogen); polyclonal goat anti-SRY-related high-mobility group box 9 (SOX9; 1:1000; # AF3075; R&D Systems); polyclonal goat anti-paired box 2 (PAX2; 1:500; # AF3364; R&D Systems); monoclonal rat anti-mCherry (1:2000; #M11217; Thermo Fisher Scientific); polyclonal rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (pERK; 1:500; #9101; Cell Signaling); anti-isolectin B4 (IB4)-biotin conjugate (1:1000; # I21414; Thermo Fisher Scientific).

Techniques: Control, Immunofluorescence

Journal: Cells

Article Title: Identification of Spinal Inhibitory Interneurons Required for Attenuating Effect of Duloxetine on Neuropathic Allodynia-like Signs in Rats

doi: 10.3390/cells11244051

Figure Lengend Snippet: NA excites the majority of AAV−NpyP + neurons via α 1B −AR. ( A ) tdTomato (tdT) expression on the fourth lumbar (L4) spinal dorsal horn (SDH) 4 weeks after microinjection of AAV−NpyP−tdT. Scale bar, 200 μm. Immunolabeling of tdT + cells (red) with the inhibitory interneuron marker PAX2 (green) or the lamina IIi−selective marker IB4 (blue). ( B ) Representative electrophysiological traces of AAV−NpyP + neurons that were depolarized and hyperpolarized by NA application (20 μM for 2 min). ( C ) Percentage of AAV−NpyP + neurons which responded to NA or NA with each antagonist selective for AR subtypes ( n = 28 cells from 15 rats for NA, 11 cells from 3 rats for NA + silodosin (α 1A −AR antagonist), 11 cells from 4 rats for NA + L−765,314 (α 1B −AR antagonist), 11 cells from 3 rats for NA + A−315456 (α 1D −AR antagonist), and 16 cells from 6 rats for NA + L−765,314 (α 1B −AR antagonist) + atipamezole (α 2 −AR antagonist). * p < 0.05, *** p < 0.001, Fisher’s exact test followed by Bonferroni adjustment. ( D ) Change in resting membrane potentials (ΔRMPs) of AAV−NpyP + neurons before and after NA in slices with or without each AR subtype−selective antagonist treatment.

Article Snippet: The sections were incubated in blocking solution for 2 h and followed by the primary antibodies: polyclonal goat anti-paired box 2 (PAX2; 1:500; AF3364, R&D Systems, Minneapolis, MN, USA) and isolectin B4 (IB4) biotin-conjugate (1:1000; I21414, Thermo Fisher Scientific, Waltham, MA, USA) for 48 h at 4 °C.

Techniques: Expressing, Microinjection, Immunolabeling, Marker, Membrane

Journal: Cells

Article Title: Identification of Spinal Inhibitory Interneurons Required for Attenuating Effect of Duloxetine on Neuropathic Allodynia-like Signs in Rats

doi: 10.3390/cells11244051

Figure Lengend Snippet: α 1B -AR in AAV−NpyP + neurons is responsible for NA−evoked depolarization. ( A ) mCherry expression on L4 SDH 4 weeks after microinjection of AAV−NpyP−mCherry−shmirAdra1B. Scale bar, 200 μm. Immunolabeling of mCherry + cells (red) with PAX2 (green) or IB4 (blue). Percentage of co−expressing cells was quantified (680 total mCherry + cells and 1805 total PAX2 + cells from 3 rats). Data shown as mean ± SEM. ( B ) Percentage of AAV−NpyP + neurons responded to NA in AAV−NpyP−shmirControl− or AAV−NpyP−shmirAdra1b−microinjected WT rats ( n = 18 cells from 4 rats for AAV−NpyP−shmirControl, and 16 cells from 5 rats for AAV−NpyP−shmirAdra1b). ** p < 0.01, Fisher’s exact test. ( C ) Changes in RMPs (ΔRMPs) of AAV−NpyP + neurons by NA in both groups.

Article Snippet: The sections were incubated in blocking solution for 2 h and followed by the primary antibodies: polyclonal goat anti-paired box 2 (PAX2; 1:500; AF3364, R&D Systems, Minneapolis, MN, USA) and isolectin B4 (IB4) biotin-conjugate (1:1000; I21414, Thermo Fisher Scientific, Waltham, MA, USA) for 48 h at 4 °C.

Techniques: Expressing, Microinjection, Immunolabeling

Journal: Scientific Reports

Article Title: Disparate effects of MMP and TIMP modulation on coronary atherosclerosis and associated myocardial fibrosis

doi: 10.1038/s41598-021-02508-4

Figure Lengend Snippet: Effect of MMP modulation on capillary to myocyte ratio in apolipoprotein E-deficient (Apoe -/- ) mice. Quantification and representative images of capillary to myocyte ratio as assessed in isolectin B4-immunohistochemically stained hearts from 36-week high-fat fed Apoe -/- mice deficient for ( A ) MMP-7, ( B ) MMP-9, and ( C ) MMP-12, in comparison to their respective Apoe single knockout controls. Capillary to myocyte ratio is presented as average/heart cross-section/mouse. Statistical significance is reported as * P < 0.05 using unpaired Students t-test. Scale bar represents 1 mm and is applicable to all panels. In A, n = 9/group; in B and C, n = 10/group.

Article Snippet: For dual immunohistochemistry, sections were first incubated for 30 min at room temperature with biotin-conjugated Griffonia Simplicifolia Isolectin B4 (IB4) (L2140; Sigma, Poole, UK) at 10 μg/mL, followed by horseradish peroxidase-labelled Extravidin (diluted 1:400 in 1% (w/v) BSA in PBS) and colour developed with SIGMA FAST ™ 3,3′-Diaminobenzidine (D4293; Sigma, Poole, UK).

Techniques: Staining, Knock-Out

Journal: Scientific Reports

Article Title: Disparate effects of MMP and TIMP modulation on coronary atherosclerosis and associated myocardial fibrosis

doi: 10.1038/s41598-021-02508-4

Figure Lengend Snippet: Effect of MMP inhibition and TIMP modulation on capillary to myocyte ratio in atherosclerotic apolipoprotein E-deficient (Apoe -/- ) mice. Quantification and representative images of capillary to myocyte ratio as assessed in isolectin B4-immunohistochemically stained hearts from ( A ) 36-week high-fat fed Apoe -/- mice treated with or without a broad-spectrum MMP inhibitor, ( B ) 36-week high-fat fed Apoe -/- mice deficient for TIMP-1 in comparison to Apoe single knockout controls, and ( C ) 18-week high-fat fed Apoe -/- mice with administration of a TIMP-2 helper-dependent (HD) adenovirus or an empty HD as control. Capillary to myocyte ratio is presented as average/heart cross-section/mouse. Scale bar represents 1 mm and is applicable to all panels. In A and B, n = 10/group; in C, n = 7–9/group.

Article Snippet: For dual immunohistochemistry, sections were first incubated for 30 min at room temperature with biotin-conjugated Griffonia Simplicifolia Isolectin B4 (IB4) (L2140; Sigma, Poole, UK) at 10 μg/mL, followed by horseradish peroxidase-labelled Extravidin (diluted 1:400 in 1% (w/v) BSA in PBS) and colour developed with SIGMA FAST ™ 3,3′-Diaminobenzidine (D4293; Sigma, Poole, UK).

Techniques: Inhibition, Staining, Knock-Out

Journal: Frontiers in Cellular Neuroscience

Article Title: Inhibitory Effects of Trifluoperazine on Peripheral Proinflammatory Cytokine Expression and Hypothalamic Microglia Activation in Obese Mice Induced by Chronic Feeding With High-Fat-Diet

doi: 10.3389/fncel.2021.752771

Figure Lengend Snippet: List of reagents (antibodies and assay kits) in the study.

Article Snippet: Biotin-conjugated isolectin B4 (IB4) , Sigma-Aldrich, Cat#L2140.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats

doi: 10.1038/s41598-020-76290-0

Figure Lengend Snippet: hADSCs and CM-hADSC increased the number of blood vessels at the edges of lesion cavities after a compressive spinal cord injury . Low-magnification micrograph photomontages of the spinal cord (horizontal sections, approximately 500 -750 μm down from the dorsal surface, identified as the lesion epicentre by GFAP reactivity) of rats treated with DMEM ( A ), CM-hADSC ( C ) and hADSC ( E ), 1 week after SCI, showing blood vessels and phagocytic cells, identified with Isolectin IB4 (red, A , C , E ), and its interaction with astrocytes, identified with anti-GFAP (green, A , C , E ), and DAPI-stained nuclei (blue, A , C , E ). Confocal images under high magnification of the boxed fields show the nervous tissue at the edges of lesion cavities ( B , D , F ). Areas marked by in yellow boxes were further amplified and shown in the red channel only to better reveal the morphologies of blood vessels. White arrows indicate blood vessels and white arrowheads indicate phagocytic cells (red round cells). White thick dotted lines delineate lesion cavities. Scalebar: A , C , E 3 mm; and B , D , F 200 μm .

Article Snippet: We used Isolectin IB4 Biotin Conjugated Antibody (1:200; Vector B-1205), and Dapi (1:4,000; Merck Millipore).

Techniques: Staining, Amplification

Journal: Scientific Reports

Article Title: Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats

doi: 10.1038/s41598-020-76290-0

Figure Lengend Snippet: hADSCs favoured maturation of the blood brain barrier. ( A – C ) Confocal images of the spinal cord (horizontal sections) of rats treated with DMEM ( A ), CM-hADSC ( B ) and hADSC ( C ), labelled with TUJ-1 (green, white-filled arrowheads), Isolectin IB4 (red, white arrows) and DAPI (blue), which recognizes young axons, endothelial and phagocytic cells, respectively. ( D , E ) High magnification confocal images of TUJ-1 immuno-labelled axonal fibers (green) growing in close contact with blood vessels (red) in hADSC treatment. Note that some axonal fibers were projected into the injury cavity ( E , white arrow). ( F ) Quantification of total length of blood vessels per tissue area (immuno-labelled with specific blood brain barrier antibody anti-SMI-71), 1 WPI. ( G , H , I ) Confocal images of the spinal cord (horizontal sections) of rats treated with DMEM ( G ), CM-hADSC ( H ) and hADSC ( I ), showing blood vessels identified with the blood brain barrier protein, SMI-71 (red, white arrows), and astrocytes, identified with anti-GFAP (green), and DAPI-stained nucleus (blue). ( J , K ) High magnification confocal images show that in CM-hADSC animals the BSB protein SMI71 pattern is punctuated ( J , white arrowheads), while it is continuous in hADSC–treated animals ( K ). ( L ) High resolution confocal image showing the close contact and interaction between GFAP-astrocyte (green) and the blood brain barrier (red) positive cells (white asterisk). White-filled arrowheads indicate axonal fibers (TUJ-1 positive), white arrows indicate blood vessels (anti-SMI-71 or IB4 positive), arrowheads with white outline indicate vascular breakpoints, white asterisk indicates astrocyte-blood vessel interaction, and thick white dotted lines delineate lesion cavities. Black asterisks in graphs indicate statistical differences between DMEM (n = 10), CM-hADSC (n = 10) and hADSC groups (n = 10). Values in graphs represent the means ± standard errors. *P < 0.05; **P < 0.01; ***P < 0.001. Scalebar: C , E , I —50 μm; J , L —20 μm.

Article Snippet: We used Isolectin IB4 Biotin Conjugated Antibody (1:200; Vector B-1205), and Dapi (1:4,000; Merck Millipore).

Techniques: Staining

Journal: Scientific Reports

Article Title: Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats

doi: 10.1038/s41598-020-76290-0

Figure Lengend Snippet: Pericytes were distributed along the blood vessels and in close contact with the vascular wall only in hADSCs treated animals. Confocal images of the spinal cord (horizontal sections) of rats treated with DMEM ( A , D , G , J ), CM-hADSC ( B , E , H , K ) and hADSC ( C , F , I , L , M , N , O ), 1WPI. ( A , B , C ) Histological sections were labelled with anti-alpha smooth muscle actin antibody (red, α-SMA), Isolectin IB4 (green, IB4) and DAPI (blue), which recognize pericytes, endothelial and phagocyte cells and nuclei, respectively. ( D , E , F ) Histological sections were immuno-labelled with pan-laminin antibody (in green), alpha smooth muscle actin antibody (red, α-SMA,) and DAPI (blue), to identify the basement membrane of the blood vessels, pericytes and nuclei, respectively. ( G , H , I ) Alternative pericyte marker, platelet-derived growth factor receptor-beta antibody (in red, PDGFR-β) and RECA-1 (green) blood vessel immunolabeling was performed to identify pericytes and endothelial cells, respectively. ( J , K , L ) Histological sections were labelled with neural/glial antigen 2 antibody (red, NG2), Isolectin IB4 (green, IB4) and DAPI (blue), to identify pericytes, endothelial and phagocytic cells and nuclei, respectively. ( M , N , O ) To certify that these vessel-associated cells were pericytes, we performed a triple staining: (1) anti-neural/glial antigen 2 (anti-NG2, red); (2) anti-α-SMA (green) and (3) Isolectin IB4 (white). The first two are considered reliable pericyte markers (anti-NG2 and/or anti-α-SMA), and the latter (IB4) identifies endothelial cells. The orthogonal view of the optical slice of confocal microscopy demonstrated the positive colocalization between anti-NG2 and anti-α-SMA ( N , O ). Arrowhead with white fill indicates blood vessels (IB4 positive), white arrows indicate pericytes (α-SMA, NG2, or PDGFR-β positive), arrowheads with white outline indicate close association between pericyte markers (anti-NG2 and/or anti α-SMA) and white dotted lines delineate lesion cavities. Scalebar: C, L: 100 μm, F, I, M: 20 μm.

Article Snippet: We used Isolectin IB4 Biotin Conjugated Antibody (1:200; Vector B-1205), and Dapi (1:4,000; Merck Millipore).

Techniques: Marker, Derivative Assay, Immunolabeling, Staining, Confocal Microscopy