Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Antibodies and markers used for immunohistochemistry.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Immunohistochemistry, Plasmid Preparation

Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Top Panel (A–C): A low-magnification transverse section (5 µm) of the mouse kidney is shown. Discrete labeling is shown for staining for aquaporin-2 ( A. AQP2, red), a marker of the collecting ducts, SK3 ( B. SK3, green), and a merger of both channels ( C. Merge, yellow-organge for co-localization of AQP2 and SK3). Labeling is apparent for SK3 in both the cortex (label C) and medullary (label M) (dashed line shows cortical-medullary demarcation). Middle Pannel (D–F): Magnified view of the yellow inset box from A. SK3 co-localizes with all AQP2-postive tubules as show by the yellow-orange images (F., asterisk). SK3 staining is also apparent in AQP2-negative structures including other tubular structures (F., arrows) and smaller secondary structures (possibly vascular structures, F., arrow heads). Bottom Panel (G–H): Magnified view of staining in the presence of SK3 blocking peptide. All SK3 staining is abolished demonstrating specificity of our anti-SK3 antibody. Scale bar is 50 µm.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Labeling, Staining, Marker, Blocking Assay

Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Section (5 µm) from WT mouse kidney showing staining for AQP2 (red), a marker of PCs in collecting duct, and SK3 (green). Panels A, C, and E are low magnification views of a cross-section through a CCD identified by AQP2 staining. Panels B, D, and F represent a magnified view of the inset area from A (yellow inset box). Panel B shows strong AQP2 staining along the luminal border of PCs (5–6 cells), but not of the ICs (2 cells without staining). As shown in D and F , strong staining of SK3 is evident along the luminal border of all cells, both PCs and ICs. Variable, but weak staining, is also apparent along the abluminal border of some cells. However, the staining is most pronounced along the luminal border for both PCs and ICs, although typically stronger in PCs, as indicated by the SK3 fluorescence line intensity profiles across (luminal to abluminal direction) two cells identified as PC and IC ( Panel G ). H . Relative mean intensity profiles (± SEM) across the cells from all sections showing the maximal values across the luminal border (Apical) and abluminal border (Basal) and the minimal values within the cytoplasm (Cytosol). The mean values are given for both PCs (n = 37) and ICs (n = 12) from all sections analyzed. The maximal luminal intensity is much greater than the abluminal intensity (*P<0.02) indicating dominant expression at the luminal border. Scale bar is 10 µm.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Staining, Marker, Fluorescence, Expressing

Journal: Microbial Cell Factories

Article Title: A fine-tuned yeast surface-display/secretion platform enables the rapid discovery of neutralizing antibodies against Clostridioides difficile toxins

doi: 10.1186/s12934-023-02200-4

Figure Lengend Snippet: Surface display and secretion of Fab-AH3/E3 in diploid Y-AH3/E3. a . The galactose induced yeast cells were harvested and stained to detect Fab display by FACS. The cells were incubated with mouse anti-HA and goat anti-human kappa followed by secondary labeling with donkey anti-mouse Dylight 550 and donkey anti-goat AF 488. The non-induced cells were used as a negative control. b. Neutralization assay with secretion culture supernatant. The yeast cells were induced to secrete Fab in SD medium containing 1% or 2% glucose at 30 °C for 72 h. The supernatants were diluted 4 times and co-cultured with Vero cells in the presence of 50 ng/mL TcdA or 10 pg/mL TcdB at 37 °C overnight. The images were taken using a phase-contrast microscope

Article Snippet: To evaluate surface display by flow cytometry, the cells were incubated with mouse anti-HA (Abcam, ab18181, 1:1000) and goat anti-human KC (Southern Biotech, 2060-01, 1:1000) followed by secondary labeling with donkey anti-mouse Dylight 550 (Invitrogen, SA5-10167, 1:500) and donkey anti-goat Alexa Fluor (AF) 488 (Invitrogen, A-11,055, 1:500).

Techniques: Staining, Incubation, Labeling, Negative Control, Neutralization, Cell Culture, Microscopy

Journal: Biomolecules

Article Title: Feasibility of Ex Vivo Ligandomics

doi: 10.3390/biom15010145

Figure Lengend Snippet: Flow cytometric analysis of the purity of immunopanned cells. Endothelial cells (ECs) and retinal ganglion cells (RGCs) were immunopanned using anti-CD31 and anti-CD90.2 mAb, respectively, detached by pipetting and analyzed by flow cytometry. ( A ) Immunopanned ECs were labeled with anti-CD31 mAb and quantified with 99.29% purity. ( B ) Human retinal microvascular endothelial cells (HRMVECs) were used as a positive control with 99.89% purity, as labeled with anti-CD31 mAb. ( C ) Immunopanned RGCs were labeled with anti-βIII-tubulin mAb and quantified with 84.80% purity. ( D ) HT22 neuronal cell line was used as a positive control with 97.87% purity, as detected by anti-βIII-tubulin mAb.

Article Snippet: To determine the purity of immunopanned cells, ECs were detached from the plates by pipetting, immunolabeled with DyLight 550-conjugated rat anti-CD31 mAb (1:100 dilution; Novus Biologicals, Centennial, CO, USA; Cat. #NB100-1642R), and incubated for 30 min at 4 °C in the dark.

Techniques: Flow Cytometry, Labeling, Positive Control

Journal: Biomolecules

Article Title: Feasibility of Ex Vivo Ligandomics

doi: 10.3390/biom15010145

Figure Lengend Snippet: Ex vivo Scg3-Phage and VEGF-Phage binding to immunopanned CD31 + ECs. Cells were isolated from the retinas of healthy (white bar) and diabetic (black bar) mice. Control phage ( n = 4 wells) was included as a negative control. ( A ) Binding of Scg3-Phage to ECs in the presence or absence of anti-Scg3 hFab ( n = 5 wells/group). ( B ) Binding of VEGF-Phage to ECs in the presence or absence of aflibercept ( n = 5 wells/group). ± SEM; ns, not significant; t -test.

Article Snippet: To determine the purity of immunopanned cells, ECs were detached from the plates by pipetting, immunolabeled with DyLight 550-conjugated rat anti-CD31 mAb (1:100 dilution; Novus Biologicals, Centennial, CO, USA; Cat. #NB100-1642R), and incubated for 30 min at 4 °C in the dark.

Techniques: Ex Vivo, Binding Assay, Isolation, Control, Negative Control

Journal: EBioMedicine

Article Title: Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis

doi: 10.1016/j.ebiom.2017.10.033

Figure Lengend Snippet: Neovascularisation synchronises neurodegeneration in EAE. (a) Representative images of spinal cord sections labeled with PKCγ, APP, or CD105. (b) Quantitative analysis of the fluorescent area of PKCγ or APP around EAE lesions. CD105 + neovessel length around EAE lesions; n = 4–6 for all experiments; error bars represent the s.e.m. ** P < 0.01, ANOVA with Tukey's multiple comparison tests. (c) Representative images of spinal cord sections labeled with CD31, Ki67, and CD105. (d) CD4 + and CD11b + cell accumulation around EAE lesions 7 days after EAE induction. Scale bars for a, d, 200 μm; c, 50 μm.

Article Snippet: The primary antibodies used were as follows: rabbit anti-mouse LDHA (1:100, PAB370Mu01; USCN), rabbit anti-mouse PKC-γ (1:100, sc-211; Santa Cruz Biotechnology, RRID:AB_632234), rabbit anti-human PKCγ (1:100, LS-C91497; LSBio, RRID:AB_2171760), rabbit anti-human LDHB (ab75167), rabbit anti-mouse NeuN (1:100, MAB377; Covance, RRID:AB_2298772), rat anti-mouse CD4 (1:100, 550,278; BD Biosciences, RRID:AB_393574), rabbit anti-mouse Iba1 (1:100, sc-98468; Santa Cruz Biotechnology), mouse anti-mouse GFAP (1:100, G3893; Sigma-Aldrich), mouse anti-APC (1:100, OP80; Calbiochem, RRID:AB_2224389), rabbit anti-mouse CD31 (1:100, ab28364; Abcam, RRID:AB_726362), rat anti-mouse CD105 (1:100, 550,546; BD Biosciences, RRID:AB_2277916), rabbit anti-amyloid β (A4) precursor protein (APP) (1:100, A8718, Sigma) and mouse anti-mouse Ki67 (1: 200, 556,003; BD Biosciences, RRID:AB_396287).

Techniques: Labeling

Journal: EBioMedicine

Article Title: Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis

doi: 10.1016/j.ebiom.2017.10.033

Figure Lengend Snippet: Axonal LDHA is required for CNS angiogenesis. (a) Double immunohistochemical labeling for NeuN and LDHA (upper panels) or LDHB (lower panels) in the cerebral cortex. (b) Double immunohistochemical staining for LDHA and PKCγ in the spinal cord. (c) Double immunocytochemical staining for LDHA and phalloidin in cultured cortical neurons. (d, e) Double immunohistochemical staining for LDHA and GFAP (d) or APC (e). (f) LDHA silencing decreases LDHA expression in the motor cortex. Expression of LDHA protein in the motor cortex of mice that underwent transfection of LDHA siRNA into the motor cortex; n = 3 each. (g) Representative images of CD105-labeled spinal cord sections obtained 7 days after EAE induction. Length of CD105 + neovessels around EAE lesions, n = 5 each. (h) Relative expression of Vegfa and Vegfb around EAE lesion with or without Lhda knock down, n = 4 each; all error bars represent the s.e.m. * P < 0.05, ** P < 0.01 relative to control, # P < 0.05 relative to lysate. ANOVA with Tukey's multiple comparison tests. Scale bars for a, c, 50 μm; b, d, e, g, 200 μm.

Article Snippet: The primary antibodies used were as follows: rabbit anti-mouse LDHA (1:100, PAB370Mu01; USCN), rabbit anti-mouse PKC-γ (1:100, sc-211; Santa Cruz Biotechnology, RRID:AB_632234), rabbit anti-human PKCγ (1:100, LS-C91497; LSBio, RRID:AB_2171760), rabbit anti-human LDHB (ab75167), rabbit anti-mouse NeuN (1:100, MAB377; Covance, RRID:AB_2298772), rat anti-mouse CD4 (1:100, 550,278; BD Biosciences, RRID:AB_393574), rabbit anti-mouse Iba1 (1:100, sc-98468; Santa Cruz Biotechnology), mouse anti-mouse GFAP (1:100, G3893; Sigma-Aldrich), mouse anti-APC (1:100, OP80; Calbiochem, RRID:AB_2224389), rabbit anti-mouse CD31 (1:100, ab28364; Abcam, RRID:AB_726362), rat anti-mouse CD105 (1:100, 550,546; BD Biosciences, RRID:AB_2277916), rabbit anti-amyloid β (A4) precursor protein (APP) (1:100, A8718, Sigma) and mouse anti-mouse Ki67 (1: 200, 556,003; BD Biosciences, RRID:AB_396287).

Techniques: Immunohistochemical staining, Labeling, Staining, Cell Culture, Expressing, Transfection

Journal: EBioMedicine

Article Title: Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis

doi: 10.1016/j.ebiom.2017.10.033

Figure Lengend Snippet: LDHA is sufficient to evoke CNS angiogenesis. (a) Representative images of CD105-labeled spinal cord sections obtained 7 days after LDHA administration. (b) Length of CD105 + neovessels around the LDHA administration site as indicated in a, n = 5 each. (c) Representative image of a Nissl-stained brain section after controlled cortical impact (CCI). (d) Representative image of the CD105-immunolabelled cerebral cortex obtained 7 days after CCI. (e) Length of CD105 + neovessels around CCI lesions as indicated in d; n = 5 each, all error bars represent the s.e.m. ** P < 0.01, Student's t -tests. Scale bars, 200 μm.

Article Snippet: The primary antibodies used were as follows: rabbit anti-mouse LDHA (1:100, PAB370Mu01; USCN), rabbit anti-mouse PKC-γ (1:100, sc-211; Santa Cruz Biotechnology, RRID:AB_632234), rabbit anti-human PKCγ (1:100, LS-C91497; LSBio, RRID:AB_2171760), rabbit anti-human LDHB (ab75167), rabbit anti-mouse NeuN (1:100, MAB377; Covance, RRID:AB_2298772), rat anti-mouse CD4 (1:100, 550,278; BD Biosciences, RRID:AB_393574), rabbit anti-mouse Iba1 (1:100, sc-98468; Santa Cruz Biotechnology), mouse anti-mouse GFAP (1:100, G3893; Sigma-Aldrich), mouse anti-APC (1:100, OP80; Calbiochem, RRID:AB_2224389), rabbit anti-mouse CD31 (1:100, ab28364; Abcam, RRID:AB_726362), rat anti-mouse CD105 (1:100, 550,546; BD Biosciences, RRID:AB_2277916), rabbit anti-amyloid β (A4) precursor protein (APP) (1:100, A8718, Sigma) and mouse anti-mouse Ki67 (1: 200, 556,003; BD Biosciences, RRID:AB_396287).

Techniques: Labeling, Staining

Journal: EBioMedicine

Article Title: Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis

doi: 10.1016/j.ebiom.2017.10.033

Figure Lengend Snippet: Vascular endothelial cell vimentin is associated with neurodegeneration-related angiogenesis. (a) Representative immuno-electron microscopy images of surface vimentin on vascular endothelial cells in the spinal cord. (b) Representative image of a spinal cord section injected with CD31-targeted liposomes containing Cy5.5 dye and the indicated oligonucleotides. Scale bars, 50 μm. (c) The graph shows the relative intensity of vimentin expression in Cy5.5 + CD31 + double-positive cells; n = 3 each, error bars represent the s.e.m. (d) Representative images of the CD105-immunolabelled spinal cord sections. (e) Length of CD105 + neovessels around EAE lesions as indicated in e. n = 5 each. (f) Correlation of vimentin expression and CD105 + neovessel length. ** P < 0.01, Student's t -tests. All error bars represent the s.e.m. ** P < 0.05, ** P < 0.01, Student's t -tests or log-rank tests. Scale bars for a, 2 μm; b, d 50 μm, e, 200 μm.

Article Snippet: The primary antibodies used were as follows: rabbit anti-mouse LDHA (1:100, PAB370Mu01; USCN), rabbit anti-mouse PKC-γ (1:100, sc-211; Santa Cruz Biotechnology, RRID:AB_632234), rabbit anti-human PKCγ (1:100, LS-C91497; LSBio, RRID:AB_2171760), rabbit anti-human LDHB (ab75167), rabbit anti-mouse NeuN (1:100, MAB377; Covance, RRID:AB_2298772), rat anti-mouse CD4 (1:100, 550,278; BD Biosciences, RRID:AB_393574), rabbit anti-mouse Iba1 (1:100, sc-98468; Santa Cruz Biotechnology), mouse anti-mouse GFAP (1:100, G3893; Sigma-Aldrich), mouse anti-APC (1:100, OP80; Calbiochem, RRID:AB_2224389), rabbit anti-mouse CD31 (1:100, ab28364; Abcam, RRID:AB_726362), rat anti-mouse CD105 (1:100, 550,546; BD Biosciences, RRID:AB_2277916), rabbit anti-amyloid β (A4) precursor protein (APP) (1:100, A8718, Sigma) and mouse anti-mouse Ki67 (1: 200, 556,003; BD Biosciences, RRID:AB_396287).

Techniques: Immuno-Electron Microscopy, Injection, Expressing