CMs were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde dissolved in PBS for 20 min. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min, washed in PBS + 0.1% Tween 20 (PBST), and blocked with 5% normal goat serum (NGS, Thermo Fisher Scientific) in PBST. The cells were stained with the following primary antibodies: anti-cTnT (Thermo Fisher Scientific), anti-α-actinin (Sigma-Aldrich), anti-MLC2v (Proteintech, Rosemont, IL, USA), anti-MLC2a (Synaptic Systems, Goettingen, Germany), and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA) antibodies at 4 °C overnight in 2% NGS in PBST. The cells were washed twice in PBST and incubated for 1 h with the following secondary antibodies: Alexa Fluor 488 anti-mouse IgG1, Alexa Fluor 594 goat anti-mouse IgG2b, and Alexa Fluor 594 goat anti-rabbit IgG (all from Molecular Probes, Eugene, OR, USA). The nuclei were stained with DAPI, and the stained cells were mounted using a fluorescent mounting solution (DAKO, Carpinteria, CA, USA). Immunofluorescence images were acquired using a fluorescence microscope (Olympus-Europa GmbH, Hamburg, Germany) and a confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Alexa fluor 488 anti mouse igg1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 anti-mouse IgG1 is a fluorescent-labeled secondary antibody used for detection and visualization of mouse IgG1 antibodies in various immunological applications. It provides a bright fluorescent signal and enables sensitive detection of target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igm alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 350 anti mouse igg2b, by Thermo Fisher Scientific (2 mentions)
Ba f8, by Developmental Studies Hybridoma Bank (2 mentions)
Sc 71, by Developmental Studies Hybridoma Bank (2 mentions)
Alexa fluor 647 anti mouse igg2a, by Thermo Fisher Scientific (2 mentions)
Confocal fluorescence microscope, by Zeiss (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti α actinin, by Merck Group (1 mentions)
Fluorescent mounting solution, by Agilent Technologies (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti mlc 2v, by Proteintech (1 mentions)
Anti ctnt, by Thermo Fisher Scientific (1 mentions)
Anti mlc2a, by Synaptic Systems (1 mentions)
Lc3b 2775, by Cell Signaling Technology (1 mentions)
Anti ubiquitin p4d1, by Cell Signaling Technology (1 mentions)
A21241, by Thermo Fisher Scientific (1 mentions)
Cy3 anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
A21121, by Thermo Fisher Scientific (1 mentions)
Mouse anti α syn, by BD (1 mentions)
10 protocols using alexa fluor 488 anti mouse igg1
1
Immunofluorescence Staining of Cardiac Markers
2
Comprehensive Antibody Catalogue for Muscle and Calcium Signaling
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Primary antibodies against PLN (2D12), phosphorylated-NFATc1 (PA5-38301), total NFATc1 (MA3-024), SERCA2a (MA3-919), ryanodine receptor 1 (RyR, MA3-925), dihydropyridine receptor α1 subunit (DHPR, MA3-920), and calsequesterin I and II (CSQ, MA3-913) were obtained from Pierce Antibodies. The primary antibody directed against SLN was generated by Lampire Biological Laboratories (PA, USA) [28 (link)]. LC3B (2775) and p62 (GP62-C) antibodies were obtained from Cell Signaling Technology (MA, USA) and Progen Biotechnik (Heidelberg, Germany), respectively. The primary antibody against actin (A2066) and stabilin-2 (orb158499) were obtained from Sigma Aldrich (MO, USA), and Biorbyt (CA, USA), respectively. SERCA1a antibody (A52) was a kind gift from Dr. David MacLennan (University of Toronto) [29 (link)]. The primary antibodies against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3) were obtained from Developmental Studies Hybridoma Bank (IA, USA). Secondary antibodies for western blotting, goat anti-mouse IgG (peroxidase conjugated) and goat anti-rabbit IgG (peroxidase conjugated) were obtained from Santa Cruz Biotechnology (TX, USA). Secondary antibodies for immunofluorescence staining, Alexa Fluor 350 anti-mouse IgG2b, Alexa Fluor 488 anti-mouse IgG1, and Alexa Fluor 555 anti-mouse IgM, were obtained from Molecular Probes (OR, USA).
3
Muscle Protein Expression Analysis
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Primary antibodies against SERCA2a (2A7-A1), PLN (2D12), dynamin 2 (PA5-19800) and RyR (MA3-925) were obtained from Pierce Antibodies. The primary antibody for SERCA1a (A52) was a kind gift from Dr David MacLennan (University of Toronto) (Zubrzycka-Gaarn et al., 1984 (link)). The primary antibody directed against SLN was generated by Lampire Biological Laboratories (Fajardo et al., 2013 (link)). Anti-ubiquitin (P4D1) and anti-nitrotyrosine (189542) antibodies were obtained from Cell Signaling Technology and Cayman Chemicals, respectively. The primary antibody against α-actin (A4700) was obtained from Sigma-Aldrich. The primary antibodies against MHCI (BA-F8), MHCIIa (SC-71), MHCIIb (BF-F3), embryonic MHC (BF-F6) (Schiaffino et al., 1989 (link); Lucas et al., 2000 (link)) and dystrophin (3B7) (Nguyen thi et al., 1990 (link)) were obtained from Developmental Studies Hybridoma Bank. Secondary antibodies for western blotting, goat anti-mouse IgG (peroxidase conjugated) and goat anti-rabbit IgG (peroxidase conjugated) were obtained from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence staining, Alexa Fluor 350 anti-mouse IgG2b, Alexa Fluor 488 anti-mouse IgG1 and Alexa Fluor 555 anti-mouse IgM, were obtained from Molecular Probes.
4
Immunocytochemistry for Neuronal Markers
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Cells were fixed with 4% paraformaldehyde
in PBS at room temperature for 15 min. Cells were blocked and permeabilized
with TBS with low triton (0.01% Triton X-100) and 3% donkey serum
for 2 h. Subsequently, cells were incubated for 48 h at 4 °C
with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660,
Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn
(1:500, 610787, BD Biosciences). Samples were then incubated with
secondary antibodies for 2 h at 37 °C: Alexa Fluor 647 anti-mouse
IgG2a (1:100, A21241, Invitrogen), Cy3 anti-rabbit IgG (1:200, 711-165-152,
Jackson), and Alexa Fluor 488 anti-mouse IgG1 (1:100, A21121, Invitrogen).
To visualize nuclei, slides were stained with DAPI (1:5000, Invitrogen)
and then mounted with PVA:DABCO. Images were taken using a Leica SP5
confocal microscope and analyzed with FIJI Is Just ImageJ. For quantification
of cytoplasmic accumulation of α-syn, the fluorescence intensity
in the green channel was corrected using the number of DAns (area
green channel/area red channel). The scoring of α-syn-positive
DAns was performed by researchers blinded to the experimental conditions.
in PBS at room temperature for 15 min. Cells were blocked and permeabilized
with TBS with low triton (0.01% Triton X-100) and 3% donkey serum
for 2 h. Subsequently, cells were incubated for 48 h at 4 °C
with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660,
Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn
(1:500, 610787, BD Biosciences). Samples were then incubated with
secondary antibodies for 2 h at 37 °C: Alexa Fluor 647 anti-mouse
IgG2a (1:100, A21241, Invitrogen), Cy3 anti-rabbit IgG (1:200, 711-165-152,
Jackson), and Alexa Fluor 488 anti-mouse IgG1 (1:100, A21121, Invitrogen).
To visualize nuclei, slides were stained with DAPI (1:5000, Invitrogen)
and then mounted with PVA:DABCO. Images were taken using a Leica SP5
confocal microscope and analyzed with FIJI Is Just ImageJ. For quantification
of cytoplasmic accumulation of α-syn, the fluorescence intensity
in the green channel was corrected using the number of DAns (area
green channel/area red channel). The scoring of α-syn-positive
DAns was performed by researchers blinded to the experimental conditions.
5
Myoblast Immunostaining and Analysis
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Immunostaining of BrdU-positive myoblasts, MyHC, and MF20 in myotubes was performed as described previously (O’Leary et al., 2013 ; Hwang et al., 2014 (link); Zhang et al., 2014 (link)). For BrdU incorporation assay, after transfection, myoblasts were incubated with 10 µg/ml BrdU (Sigma-Aldrich) for 2 h before harvest. For MyHC and MF20 staining, after transfection, myoblasts were subjected to myogenic differentiation for 2 days. Cells were fixed with cold methanol, and then blocked with 3% bovine serum albumin in PBS for 1 h, followed by incubation with primary antibodies for BrdU (Santa Cruz), MyHC (Santa Cruz), and MF20 (DSHB) for 2 h. Alexa Fluor 488 anti-mouse IgG1 (Invitrogen) or Alexa Fluor 594 anti-rabbit IgG1 (Invitrogen) was added as secondary antibodies for 1 h at room temperature.
6
Immunoblotting, Immunoprecipitation, and Immunohistochemistry Antibodies
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The antibodies used for immunoblotting, immunoprecipitation and immunohistochemistry were: PERM1 (Sigma, #HPA031711), diluted 1:1,000 for immunoblotting, 1:100 for immunohistochemistry; BNIP3 (Cell Signaling, #3769), diluted 1:1,000; TOM20 (Abcam, #ab56783), diluted 1:100 for immunohistochemistry; TOM20 (Sigma, #HPA011562), diluted 1:5,000 for immunoblotting, 1:100 for immunohistochemistry; GAPDH (Invitrogen, #AM4300), diluted 1:10,000 for immunoblotting; Ankyrin B (Invitrogen, #33-3700), diluted 1:1000 for immunoblotting, 1:100 for immunohistochemistry; MYH7 (Developmental Studies Hybridoma Bank [DSHB], BA-F8), MYH2 (DSHB, SC-71), MYH4 (DSHB, BF-F3), CD31/PECAM (BD Pharmingen, #553370), all diluted 1:100 for immunohistochemistry; anti-FLAG M2-HRP (Sigma, #A8592), anti-HA-HRP (Miltenyi, #130-091-972), Alexa Fluor 350 anti-mouse IgG2b (Life Technologies, #A-21140), Alexa Fluor 488 anti-mouse IgG1 (Life Technologies, #A-21121), Alexa Fluor 546 anti-mouse IgM (Life Technologies, #A-21045), Alexa Fluor 546 anti-rabbit (H+L) (Life Technologies, #A-11010), Alexa Fluor 488 anti-rat (Life Technologies, #A-11006), anti-mouse HRP (Sigma, #A9044) and anti-rabbit HRP (Sigma, #A0545), all diluted 1:4,000 for immunoblotting and 1:200 for immunohistochemistry.
7
Measuring DNA Fiber Replication Dynamics
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maRTA was performed as previously described with some modifications [31 (link)]. Briefly, 36 hours after siRNA transfection, HeLa cells were pulse-labeled with 50 μM iododeoxyuridine (IdU) for 40 min. Cells were then treated or not with 2 mM HU for 5 hours or 16 hours. The cells were released in fresh medium containing 50 μM of chlorodeoxyuridine (CldU) for 40 min. Cells were then harvested and embedded into agarose plugs containing 20,000 cells/plug. After proteinase K digestion and agarose digestion by beta-agarase, DNA fibers were stretched on 3-aminopropyltriethoxysilane coated slides (LabScientific) using polydimethylsiloxane molds fashioned with micro-capillary channels prepared as described [31 (link)]. DNA fibers were then denatured in 2.5 M HCl, and probed with the following antibodies: mouse IgG1 anti-BrdU/IdU (clone BD44, Becton Dickinson), rat anti-BrdU/CldU (clone B1/75, Bio-Rad OBT0030), and mouse IgG2a anti-ssDNA (clone 16–19, Millipore). Secondary antibodies included Alexa Fluor 488 anti-mouse IgG1, Alexa Fluor 594 anti-rat, and Alexa Fluor 647 anti-mouse IgG2a, respectively (Life Technologies). Images were acquired on Leica DMI6000 epifluorescence microscope using Leica LAS-AF software. Signals were measured using NIH ImageJ software with custom-made modifications and the data analyzed with GraphPad Prism software.
8
Organoid and BMSC Staining Protocol
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For organoid staining, cells were plated in 5% Growth Factor Reduced Matrigel Matrix (Corning) and 3 to 5 d later were harvested in cold PBS. Organoids were then washed with 1X Carbo-Free Blocking Solution (Vector Laboratories) for 5 min and stained using biotin-StcEE447D (34 (link)) in PBS for 1 h on ice. After a wash with PBS, cells were stained with Streptavidin-647 (Life Technologies, 1:1,000) for 20 min at room temperature and washed again with PBS. Cells were then resuspended in a drop of Vectashield (Vector Laboratories) to mount on glass slides and image right away using the Nikon ECLIPSE Ti2-E fluorescence microscope. Quantifications were done using imageJ where MFI was calculated for manually surrounded organoids. For BMSC staining, cells were plated on collagen-coated glass slides, fixed with 4% PFA, permeabilized with 0.1% Triton-X 100 in PBS and blocked with 1% BSA-PBS before incubation with primary antibodies [anti-PDGFRβ (BioRad 7460-3104, 1:200) and anti-αSMA (Fisher, MS113P, 1:500)] overnight. After washes with PBS, slides were incubated with secondary antibody for 30 min (Life Technologies, Alexa Fluor 647 anti-Mouse IgG2a, Alexa Fluor 488 anti-Mouse IgG1, 1:500) and imaged as described.
9
DNA Fiber Assay for Replication Dynamics
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Exponentially growing cells were pulsed with 30 μM CldU (Merk-Milipore) for 30 min and then with 250 μM IdU (Merk-Milipore) for another 30 min. Cells were then lysed on the slide by adding spreading buffer (0.5% SDS, 200 mM Tris pH 7.4, 50 mM EDTA) and incubated for 6 min at RT in humidity chamber. DNA fibers were stretched by tilting the slide ∼30° and, after air drying, fixed for with ice-cold 3:1 methanol:acetic acid solution. Slides were then incubated in 2.5 HCl for 30 min at RT, and blocked with PBS + 1% BSA + 0.1% Triton X-100 before incubation with anti-CldU (anti-BrdU, sc-56258 BU 1/75 ICR1 from Santa Cruz Biotechnology, RRID: AB_305426) and anti-IdU (anti-BrdU, B44 from BD Bioscience, RRID:AB_2313824 ) antibodies overnight at 4°C. The slides were then incubated with anti-rat IgG Alexa Fluor 555 (Thermo Fisher Scientific) and anti-mouse IgG1 Alexa Fluor 488 (Thermo Fisher Scientific). Finally, they were incubated with anti-ssDNA (MAB3034 from Merck Millipore) and anti-mouse IgG2a Alexa Fluor 647 (Thermo Fisher Scientific) and mounted with Prolong (Thermo Fisher Scientific). Visual acquisition of the DNA fibers was done with a Zeiss Cell Observer fluorescent microscope equipped with a 40× NA 1.3 oil immersion objective and ZEN imaging software. Images were analyzed with the image processing program FIJI software (36 (link)).
10
Evaluating HUVEC Viability and Phenotype
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Human Umbilical Endothelial Cells (HUVECs, Gibco) were cultured in Medium 200 (MÀ200, Gibco) supplemented with Large Vessel Endothelial Supplement (LVES), at 37 °C with 5 % of CO 2 . Upon confluence of 80 %, HUVECs were trypsinized and cultured on the coated substrates at a density of 5000 cells/cm 2 (for evaluation of cell morphology and adhesion) or 50,000 cells/cm 2 (for evaluation of reendothelization) using Low Serum Growth Supplement (LSGS, Gibco). After 24 h of culture, cell viability was quantified with Alamar Blue (BIO-RAD) following the manufacturer's instructions. The substrates were then washed with PBS and fixed with 10 % formalin for the following immunocytochemical analysis. The expression of von Willebrand factor (vWF), a key endothe-lial marker, was visualized by staining with anti-vWF antibody (0.5 mg/mL, ab201336, abcam) and secondary antibody antimouse IgG1 (Alexa Fluor 488, 1:400, ThermoFisher Scientific). Nuclei were stained with DAPI (1:500) and actin filaments with phalloidin (1:50). Images were taken with a confocal laser scanning microscope (TCS SP8, Leica). Cell morphology analysis was performed with Fiji (Image J). TCPS was used as a control substrate.
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