Matrigel was purchased from BD Biosciences (San Jose, CA). Monoclonal anti-NFκB-p65, anti-actin and horseradish peroxidase (HRP)-linked antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, MA). Propidium iodide and Alexa 568-conjugated goat anti-mouse IgG were from Molecular Probes (Eugene, OR) and DRAQ-5 from Alexis Biochemicals, (San Diego, CA). 2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3 penta-fluoropropyl) acetamide (EF5) and antibody detecting EF5 were generous gifts from Dr. Cameron J. Koch (University of Pennsylvania, School of Medicine, USA). Recombinant human NFκB-p65 (12–317, untagged) was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). In situ cell death detection kit based on the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method was from Roche Diagnostics (Indianapolis, IN). OCT embedding medium was from Leica Biosystems (Buffalo, IL). PABA/NO was prepared as previously described [26 (link)]. NAC, GSH, GSSG reductase (yeast) and all other chemicals were purchased from Sigma (St. Louis, MO).
Alexa 568 conjugated goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568-conjugated goat anti-mouse IgG is a secondary antibody used for fluorescent detection of mouse primary antibodies in various immunoassays and imaging techniques. It consists of a goat-derived anti-mouse IgG antibody labeled with the Alexa Fluor 568 fluorescent dye.
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Lab products found in correlation
Alexa 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (5 mentions)
Alexa 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti neun, by Merck Group (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Horseradish peroxidase hrp linked antibodies, by Santa Cruz Biotechnology (1 mentions)
Draq 5, by Alexis Biochemicals (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Matrigel, by BD (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Alexa 568 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated goat antirat igg, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Lsm digital image analyzer, by Zeiss (1 mentions)
Anti flag, by Merck Group (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Lsm 700 meta confocal microscope, by Zeiss (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
14 protocols using alexa 568 conjugated goat anti mouse igg
1
Evaluating NFκB-p65 Pathway Activation
2
Immunohistochemical Profiling of Neural Markers
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A mouse monoclonal antibody against GM2 ganglioside (GMB28; immunoglobulin M, 1:20) was kindly donated by Dr. Tai (Department of Tumor Immunity, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). An anti-GFAP (Dako, Carpinteria, CA, USA, 1:1000), anti-Iba1 (Wako Pure Chemical Industries, Osaka, Japan, 1:500), anti-CD68 (clone FA-11, AbD Serotec Ltd., Oxford, UK, 1:100), anti-NeuN (EMD Millipore, Billerica, MA, 1;1000) and anti-S100ß (GeneTex, Irvine, CA, 1:100) were used as primary antibodies. As secondary antibodies, Alexa-488-conjugated goat anti-mouse IgG (1:1000), Alexa-568-conjugated goat anti-mouse IgG (1:1000), Alexa-568-conjugated goat anti-mouse IgM (1:1000), Alexa-488-conjugated goat anti-rat IgG (1:1000), Alexa-488-conjugated goat anti-rabbit IgG (1:1000), Alexa-568-conjugated goat anti-rabbit IgG (1:1000) (all purchased from Molecular Probes, Eugene, OR, USA), and Histofine Simple Stain MAX-PO(R) (Nichirei Co., Tokyo, Japan) were used.
3
Immunofluorescence Staining of Transfected Cells
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MN9D cells cultured on 100 μg/mL poly-d -lysine-coated aclar film (Electron Microscopy Science, Washington, PA, USA) were placed in a 4-well culture plate (Nunc, Roskilde, Denmark) and transiently transfected with the indicated plasmids. Twenty-four-hours post-transfection, cells were washed with PBS and fixed with either 4% paraformaldehyde at room temperature or 100% methanol at −20 °C for 20 min. Primary hippocampal neurons were fixed with 4% paraformaldehyde at room temperature for 20 min. The cells were then blocked for 1 h in PBS containing 5% normal goat serum and 0.1% Triton X-100, followed by incubation at 4 °C overnight with anti-V5 (1:200; Invitogen), anti-FLAG (1:200), or anti-NeuN (1:200, Millipore) antibodies in PBS containing 1% normal goat serum and 0.1% Triton X-100. After several washes with PBS, fluorescent secondary antibodies were used: Alexa 568-conjugated goat anti-mouse IgG (1:200; Molecular Probes, Eugene, OR, USA) or Alexa 488-conjugated goat anti-rabbit IgG (1:200; Molecular Probes). Nuclei were counterstained with 1 mg/mL Hoechst 33258 (Molecular Probes), and cells were examined under an LSM 700 META confocal microscope equipped with epifluorescence and an LSM digital image analyzer (Carl Zeiss, Zena, Germany). For microscopic examinations, we were blinded to the group allocation during experiments, data collection and assessing outcome.
4
Double-Label Immunofluorescence of αsyn and Tau
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For double-label immunofluorescence to detect phosphorylated αsyn and tau, brain sections were incubated overnight at 4°C in a cocktail of #64 antibody and anti-pS396 antibody. The sections were washed and incubated in a cocktail of Alexa568-conjugated goat anti mouse IgG (Molecular Probes) and Alexa488-conjugated goat anti rabbit IgG (Molecular Probes). After further washing, sections were stained with TOPRO-3, coverslipped with Vectashield (Vector) and observed with a laser-scanning confocal fluorescence microscope (LSM5 PASCAL; Carl Zeiss).
5
Detailed Immunofluorescence Staining Protocol
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The procedure for immunofluorescence staining and image collection has been described in detail before (8 (link)). For visualization of adhesion molecules we used the following monoclonals: DSG1-P23 and DSG1-P124 (DSG1 ectodomain), 27B2, DG3.10, 18D4 and B-11 (DSG1 endodomain), DSG-G194 (DSG3), U100 (DSC1), U114 (DSC3), 15F11 (PG), DP2.15 (DP), and PKP3-270.6.2 (PKP3). Early endosomal antigen (EEA1) was stained with 14/EEA1, cathepsin D (CTS D) was stained with CTD-19, LAMP-1 with H4A3, and connexin 43 with 4E6.2. Double staining of IgG and adhesion molecules IgG was performed with fluoresceinthiocyanate (FITC)-conjugated Fcγ-specific goat F(ab')2 anti-human IgG (Protos Immunoresearch, Burlingame, CA, U.S.A) and Alexa 568-conjugated goat anti-mouse IgG (Molecular Probes Eugene, OR, U.S.A) as secondary steps. For double staining with two different mouse monoclonals we used Zenon® Mouse IgG Labeling Kits Alexa Fluor®488 and Alexa Fluor®568 (Molecular Probes, Invitrogen, USA) by following technical protocols. Supplementary Table 1 contains details on antibodies used.
6
Endothelial Cell Inflammatory Activation Assay
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Confluent layers of endothelial cells were seeded and cultured in 96-well plates for one day. The monolayer of cells was scratched 24, 6, or 2 h before fixation. Some wells were treated with 0.6 μM of rMASP-1. Cells were fixed in ice-cold methanol–acetone (1:1) for 10 min. Then, cells were stained with primary anti-human antibodies against E-selectin, ICAM-1 or VCAM-1 (as indicated in Table 1 ) followed by Alexa568-conjugated goat anti-mouse IgG (1:500) and Hoechst 33342 (1:50,000, Invitrogen). Images were taken using an Olympus IX-81 fluorescence microscope.
7
Dual Immunocytochemical Labeling Protocol
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For a double immunocytochemical labeling, cells were fixed with 4% paraformaldehyde (EMS, Hatfield, PA, USA) at RT for 10 min at various time periods after drug treatment. For the images of TRAP1 and cytochrome c, cells were permeabilized with 0.3% saponin (Sigma) at RT for 10 min followed by blocking in 0.2% cold water fish gelatin/0.5% BSA in PBS (PBG) at RT for 30 min. After permeabilization and blocking steps, cells were incubated with a mouse anti-TRAP1 antibody, mouse anti-cytochrome C antibody (BD Biosciences; 1:200) or rabbit anti-Tom20 (Santa Cruz; 1:200) in 0.2% PBG overnight at 4℃. After extensive washing, cells were incubated with Alexa488-conjugated goat anti-rabbit IgG (Invitrogen; 1:200) and Alexa568-conjugated goat anti-mouse IgG (Invitrogen; 1:200) at RT for 1 hr. Hoechst 33258 staining (Molecular Probes; 1 µg/ml, Eugene, OR, USA) was performed for 10 min at RT. Slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Samples were observed under an LSM 510 META confocal laser scanning microscope equipped with epifluorescence and a digital image analyzer (Carl Zeiss, Zena, Germany).
8
Characterizing Cryptococcus Capsule Composition
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The reaction was modified from elsewhere [48 (link)]. An inoculum of C. neoformans was treated with r-javanicin-Flu-P1 at MIC value (25 µg/mL) and further incubation at 37 °C for 2 h and 8 h. The fungal suspension in each time point was collected and washed three times with PBS. After centrifugation at 5000× g for 5 min, yeast cells were then fixed with 4% paraformaldehyde. Fixed cells were blocked with PBS containing 2% human AB serum and further incubated on ice for 30 min. Monoclonal antibody (mAb) against glucoronoxylomannan (GXM) clone 18B7 (1:50) (kindly provided by Assoc. Prof. Dr. Sirida Youngchim, Department of Microbiology, Faculty of Medicine, Chiang Mai University, Thailand) was added and incubated on ice for 30 min. After washing, the mixture was further incubated with a 1:200 dilution of Alexa 568-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) followed by 20 µg/mL calcofluor white (CFW) (Merck KGaA, Darmstadt, Germany) for 30 min. Finally, the cell was pelleted and resuspended with anti-fade and observed under a fluorescent microscope and CLSM model LSM 980 (Carl Zeiss, Inc., Oberkochen, Germany). Control was done in parallel whereas r-javanicin-Flu-P1 was replaced by peptide suspension buffer.
9
Immunofluorescence Analysis of β-catenin
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E10 cells were cultured on 24-well plastic plates. Before collection, the cells were briefly washed with PBS and fixed with 4% paraformaldehyde for 15 min. After being washed with PBS, the cells were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 10% FBS for 1 h at room temperature. After rinsing, the cells were incubated overnight with primary antibodies against β-catenin (1 : 200). Subsequently, cells were washed with PBS and incubated with Alexa 568-conjugated goat anti-mouse IgG (Invitrogen) for 1 h. Images were acquired using a Nikon Eclipse TE-2000 inverted fluorescence microscope (Nikon Instruments, Melville, NY, USA).
10
Caco-2 Cell Immunostaining for NHE3 and Rab
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