Extracted DNA (see Section 2.3 , final concentration 10 ng/μL) and Lipofectamine were used for the introduction, according to the protocol provided by the manufactures. Validation of the cfDNA introduction was determined by immunostaining against dsDNA. Granulosa cells were cultured on a glass chamber (Millicell, Merck Millipore) in 199 medium (Thermo Fisher) containing 5% FCS and the cfDNA‐Lipofectamine mix for 24 h, then subjected to immunostaining. Immunostaining was conducted as previously described.13 Antibodies used for the immunostaining were mouse anti‐dsDNA (1:200; ab27156) and goat anti‐mouse IgG Alexa Fluor 488 (1:500; Cell Signaling). Granulosa cells were observed under a fluorescence microscope (Leica DMI 6000B, Leica). COCs were incubated in IVM medium containing the cfDNA‐Lipofectamine mix for 44 h, and oocytes were activated to determine rate of development to the blastocyst stage.
Alexa fluor 488 goat anti mouse igg
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 488 goat anti-mouse IgG is a fluorescently labeled secondary antibody. It is designed to detect and label mouse immunoglobulin G (IgG) in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 555 goat anti rabbit igg, by Cell Signaling Technology (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Cell Signaling Technology (2 mentions)
Dmi6000b, by Leica (1 mentions)
Goat anti rabbit igg alexa fluor plus 647, by Thermo Fisher Scientific (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Clathrin, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Axioplan 2 fluorescence microscope, by Zeiss (1 mentions)
Anti 53bp1, by Novus Biologicals (1 mentions)
Yh2ax, by Novus Biologicals (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin edta, by GE Healthcare (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Igg elution buffer, by Thermo Fisher Scientific (1 mentions)
Dodecyltrimethylammonium chloride, by Merck Group (1 mentions)
12 protocols using alexa fluor 488 goat anti mouse igg
1
Granulosa Cell Transfection and Oocyte Development
2
Antibody Staining for Endocytic Markers
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The primary antibodies used in this study were either made in house or were purchased from Cell Signaling Technologies. Stabilin-2 primary antibody was made by the Dr. Paul Weigel lab and was labeled as mAb30,63 (link) and Stabilin-1 primary antibody (clone 911) was a kind gift from Dr. Marko Salmi (University of Turku, Turku, Finland). Anti-mouse immunoglobulin G (IgG) Fab2 Alexa Flour 488 (Cell Signaling Technology, catalog #4408S) was used for detecting these primary antibodies. Primary antibodies for clathrin (catalog #2410S), caveolin 1 (catalog #3238S), early endosomal antigen (EEA1) (catalog #2411S), Rab7 (catalog #9367S), and LAMP1 (catalog #9091S) were purchased from Cell Signaling Technology. Goat anti-rabbit IgG Alexa Fluor Plus 647 (Invitrogen, #A32733) and goat anti-mouse IgG Alexa Fluor 488 (Cell Signaling Technologies, #4408S) secondary antibody was used for detecting these primary antibodies.
3
Quantitative DNA Damage Imaging Assay
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Cells were seeded on culture slides. Cells were pulse labeled with 10 µM EdU for 20 minutes prior to treatment. After treatment the cells were fixed, permeabilized and blocked. Foci were detected using anti-53BP1 (Rabbit-anti 53BP1, 1:2000, Novus Biologicals), RPA (Mouse-anti RPA, 1:400, Santa Cruz), yH2AX (Rabbit-anti yH2AX, 1:250, Novus Biologicals), RAD51 (Rabbit, 1:500, Calbiochem), IFN-ß1 (Rabbit-anti IFN-ß1, 1:1000, Cell signaling) or IRF3 (Rabbit-anti IRF3, 1:400, Cell Signaling) followed by Alexa Fluor 488 goat anti rabbit IgG (Cell Signaling, 1:600), AlexaFluor 488 goat anti mouse IgG (Cell signaling, 1:500), AlexaFluor 594 goat anti rabbit IgG (Abcam, 1:600) or AlexaFluor 647 goat anti rabbit IgG (Cell Signaling, 1:600) and mounted (Vector Laboratories). EdU was stained with Alexa Fluor Azide 594 (Life Technologies, 1:500) and nuclei were stained with DAPI. Foci and fluorescence Intensity were quantified manually by capturing fluorescence images using a Zeiss Axioplan 2 fluorescence microscope equipped with a charge-coupled device camera and Axiovision software followed by quantification by Image J software. RPA/yH2AX-Foci were quantified automatically by the Aklides®-system (Medipan). Foci and fluorescence intensities of 100 cells per dose per slide and experiment were quantified.
4
Immunofluorescence Microscopy of Cell Markers
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The overnight cell growth on coverslips at 4°C was followed by a 1h blocking incubation with 5% milk, then incubation with the following antibodies, all diluted at 1:100: DEPDC1, Flag and FOXM1. Anti-V5 primary antibody from Cell Signaling was used at 1:50 dilution. 4’,6-diamidino-2-phenylindol (DAPI) and either Alexa Fluor 555 goat anti-mouse Ig G or Alexa Fluor 488 goat anti-mouse IgG (both diluted 1:1000, Cell Signaling Technology, Massachusetts, USA)) were subsequently applied to the coverslips. The TCS SP5 confocal microscope from Leica Microsystems (Germany) was used to obtain cell photographs.
5
Multiplexed Influenza A Detection Using Quantum Dots
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Core/shell CdSe/ ZnS QDs with emission peaks centred at 525, 625 and 705 were purchased from Wuhan Jiayuan Quantum Dots Co. Ltd. (China, www.qds.net.cn ), and conjugated with protein A due to the action of the coupling agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Mouse-MAb to influenza A H1N1, H3N2 and H9N2 hemagglutinin (HA) were purchased from Sino Biological Inc., (China, www.sinobiological.com ). Alexa Fluor® 488 goat anti-mouse IgG was purchased from Cell Signalling Technology (USA, www.cellsignal.com ), whereas mouse-MAb anti-adenovirus was from Abcam, (UK, www.abcam.com ). Other reagents included the following: Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS), trypsin-EDTA, 0.25% solution, penicillin-streptomycin 10,000 U/ mL and Dulbecco’s phosphate buffered saline (DPBS) modified without Ca2+ and Mg2+, from GE healthcare life science (USA, www.gelifesciences.com ); 16% formaldehyde solution (methanol-free solution), IgG elution buffer (pH = 2.8), and Triton X-100 in H2O from Thermo Scientific, (USA, www.thermoscientific.com ); dodecyltrimethyl-ammonium chloride (DTAC), sodium dodecyl sulphate (SDS) and bovine serum albumin (BSA) Sigma-Aldrich (USA, www.sigmaaldrich.com ); and 5% (wt/vol) casein (alkali-soluble) Novagen, (Germany, www.novagen.com ); Hoechst 33,342, from Beyotime Biotechnology (China, www.beyotime.com ).
6
Immunodetection of HBcAg and Apoptosis in Liver
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For the immunohistochemical detection of HBcAg, liver tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Sections with a thickness of 4 μm were incubated with an anti-HBcAg antibody (Dako, Glostrup, Denmark, B0586) followed by immunoperoxidase staining using the DAB Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA). For immunofluorescence staining of HBcAg and cleaved caspase-3, the tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Sections with a thickness of 4 μm were incubated with an in-house mouse monoclonal anti-HBcAg antibody (diluted 1:100) and an anti-cleaved caspase-3 rabbit antibody (diluted 1:300, Cell Signaling Technology, Danvers, MA, USA) as primary antibodies and Alexa Fluor 488 goat anti-mouse IgG (diluted 1:500, Cell Signaling Technology) and Alexa Fluor 555 goat anti-rabbit IgG (diluted 1:500, Cell Signaling Technology) as secondary antibodies. We used VECTASHIELD mounting medium with DAPI (Vector Laboratories) for mounting. The Apoptag® Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA) was used for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of mouse liver sections according to the manufacturer’s instructions.
7
Immunofluorescence Staining of GRIK3, E-cadherin and Cadherin
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The cells were seeded on coverslips laid in six‐well plates and cultured at 37°C in 5% CO2 for 12 hours. Before staining, the cells were fixed with 4% paraformaldehyde for 30 minutes, washed three times with PBS, and permeabilized through incubation with 0.25% Triton X‐100 (Biyuntian) solution. The cells were incubated with 10% goat serum for 15 minutes at room temperature to block nonspecific binding. The cells were incubated with primary antibodies against GRIK3, E‐cadherin and cadherin (1:100, Cell Signaling Technology) overnight at 4°C. Subsequently, the cells were incubated with Alexa Fluor 488 goat antimouse IgG (Cell Signaling Technology) or Alexa Fluor 588 goat antimouse IgG (Cell Signaling Technology for 1 hour at room temperature. DAPI (Biyuntian, China) was used to stain the cell nuclei. Fluorescence images were captured by a laser‐scanning confocal microscope (Olympus FV1200MPE). The expression levels of proteins determined by the intensity of fluorescence were calculated by Image‐Pro Plus 6.0 software.
8
Temporal Immunofluorescence Analysis of BBBD in Mice
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The mice were sacrificed at 1 h, 6 h, 12 h, 24 h, and 48 h after BBBD (n = 2 per time point). To perform the immunofluorescence assay, mice were transcardially perfused with 0.9% NaCl and ice-cold 4% formaldehyde. Extracted brains were post-fixed with 4% formaldehyde for 3 days at 4 °C. Fixed brains were sliced into 30 μm sections using a vibrating blade microtome (Leica VT1200S, Leica Microsystems, Wetzlar, Germany). Tissue slices were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The tissues were incubated for 2 h in a blocking solution containing 10% normal goat serum (Abcam, Cambridge, MA, USA) followed by overnight incubation with specific primary antibodies, including rabbit anti-Iba‐1 (Wako, Osaka, Japan, #019-19741; 1:250) and mouse anti-GFAP (Sigma-Aldrich, #G3893; 1:1000). Secondary antibodies included Alexa Fluor 555 goat anti-rabbit IgG (Cell Signaling, #4413; 1: 1000), and Alexa Fluor 488 goat anti-mouse IgG (Cell Signaling, #4408; 1: 1000). The slides were mounted with a fluorescence mounting medium (Dako, Glostrup, Denmark).
9
Immunolabeling of Hippocampal Neurons in Rat Brain
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The PND30–35 rats were deeply anesthetized with sevoflurane and transcardially perfused
with phosphate buffered saline (PBS) (pH 7.4) followed by 4% formaldehyde (pH 7.4). The
hippocampus was collected and postfixed with the same fixative for 24 h at 4 °C. The
hippocampus was cut into a thickness of 30 μm coronal sections on a freezing microtome.
The sections were blocked in 3% bovine serum albumin (BSA) and 0.3% Triton X-100
(Sigma-Aldrich) for 1 h at room temperature followed by incubation with primary antibodies
as required (mouse anti-SK2 (1:1000 Millipore,); rat anti-NeuN (1: 1000, Abcam)) in 1% BSA
overnight at 4 °C. After the sections were washed, they were incubated for 1 h with
secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG, 1:500; Alexa Fluor 594 goat
anti-rabbit IgG, 1:500; both from Cell Signaling Technology) and 4′,
6-diamidino-2-phenylindole (DAPI) solution for 10 min at 37 °C. Fluorescence was detected
using a confocal laser microscope (LSM 800, Zeiss). The optical density of neuropeptide Y
(NPY) was measured using ImageJ software (National Institutes of Health).
with phosphate buffered saline (PBS) (pH 7.4) followed by 4% formaldehyde (pH 7.4). The
hippocampus was collected and postfixed with the same fixative for 24 h at 4 °C. The
hippocampus was cut into a thickness of 30 μm coronal sections on a freezing microtome.
The sections were blocked in 3% bovine serum albumin (BSA) and 0.3% Triton X-100
(Sigma-Aldrich) for 1 h at room temperature followed by incubation with primary antibodies
as required (mouse anti-SK2 (1:1000 Millipore,); rat anti-NeuN (1: 1000, Abcam)) in 1% BSA
overnight at 4 °C. After the sections were washed, they were incubated for 1 h with
secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG, 1:500; Alexa Fluor 594 goat
anti-rabbit IgG, 1:500; both from Cell Signaling Technology) and 4′,
6-diamidino-2-phenylindole (DAPI) solution for 10 min at 37 °C. Fluorescence was detected
using a confocal laser microscope (LSM 800, Zeiss). The optical density of neuropeptide Y
(NPY) was measured using ImageJ software (National Institutes of Health).
10
Immunohistochemical and Immunofluorescence Analysis of HIF-1α and HIF-2α
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The slides were dried, dewaxed, rehydrated, and blocked. Next, the sliders were boiled and then incubated with the anti-HIF-1α antibody (1:500, ProteinTech Group, Inc.) and anti-HIF-2α antibody (1:500, ProteinTech Group, Inc.) in a moist chamber. After 3 washes, the slides were incubated with a secondary antibody (Envision, Dako, Glostrup, Denmark) and then stained with DAB (3,3-diaminobenzidine). Nuclei were stained with hematoxylin. The cutoff value (45% positive cells) of HIF-1α/ HIF-2α immunoreactivity was determined by receiver operator curve (ROC) analysis. For immunofluorescence, slides were incubated with rabbit anti-E- cadherin (1:200, Abcam) and mouse anti-vimentin (1:200, Abcam) for 2 hours. Slides were incubated for 1 hour with Alexa Fluor 488 goat anti-mouse IgG (1:800, Cell Signaling Technology) and Alexa Fluor 594 goat anti-rabbit IgG (1:800, Cell Signaling Technology).
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