Primary antibodies used in this study included mouse anti-TH (Millipore, Temecula, CA, MAB318), rabbit anti-GAPDH (Cell Signaling, Danvers, MA, 2118), rabbit anti-4-HNE (Alpha diagnostics, San Antonio, TX, HNE11-S), rabbit anti-HO-1 (Millipore, Temecula, CA, AB1284), rabbit anti-pJNK (Cell Signaling, Danvers, MA, 9251), rabbit anti-JNK (Cell Signaling, Danvers, MA, 9252). Secondary antibodies used in this study included anti-mouse/rabbit HRP-linked secondary antibody (Cell Signaling, Danvers, MA, 7076 or 7074), Alexa Fluor 488 donkey anti-mouse secondary antibody (Life technologies, Eugene, OR, A21202), Alexa Fluor 568 donkey anti-rabbit secondary antibody (Life technologies, Eugene, OR, A10042), goat anti-mouse or goat anti-rabbit (Millipore, Temecula, CA, AP124 or AP132), and mouse or rabbit PAP antibody (Jackson Immunoresearch, West Grove, PA). PQ dichloride hydrate (Sigma, St. Louis, MO, 856177) was purchased and dissolved in saline for mice treatment.
Alexa fluor 488 donkey anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 donkey anti-mouse secondary antibody is a fluorescently-labeled secondary antibody. It is designed to bind to mouse primary antibodies, allowing for detection and visualization of target proteins in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Spa 810, by PhosphoSolutions (2 mentions)
H00056339 b01p, by Merck Group (2 mentions)
Hpa038002, by Santa Cruz Biotechnology (2 mentions)
Sc 374280, by Merck Group (2 mentions)
Alexa fluor 546 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Abe572, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rabbit anti jnk, by Cell Signaling Technology (1 mentions)
Rabbit anti p jnk, by Cell Signaling Technology (1 mentions)
Anti mouse rabbit hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti 4 hne, by Alpha Diagnostic (1 mentions)
Pq dichloride hydrate, by Merck Group (1 mentions)
Mouse anti th, by Merck Group (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
25g needle, by Terumo (1 mentions)
J61899, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
22 protocols using alexa fluor 488 donkey anti mouse secondary antibody
1
Protein Expression and Oxidative Stress
2
Keratin 10 Expression Analysis in NIKS Cells
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A modified protocol was used based on previous studies 23 . In brief, NIKS cells were first collected by trypsinization and after centrifugation and washing, cells were resuspended to ∼1 × 106 cells/ml in F medium and fixed in 1.5% PFA (J61899; Alfa Aesar, Lancaster, UK) for 10 min at room temperature. Cells were then recovered by centrifugation (3000 rpm) and permeabilized in ice‐cold methanol (1 × 106 cells/500 µl) (Sigma, Haverhill, UK) at 4 °C for 10 min. Cells were subsequently washed in PBS–1% bovine serum albumin (BSA) and passed gently through a 25G needle (Terumo, Leuven, Belgium) for to five times to avoid the formation of cell clumps. Cells were then incubated (1 × 106 cells/100 µl) with a mouse primary antibody to keratin 10 (Krt10) (PA5‐32459; Thermo Scientific, East Grinstead, UK) for 1 h on ice with occasional agitation. The optimal concentration of Krt10 antibody for use in these assays was determined experimentally to be 1 µg/µl. Cells were subsequently washed in PBS–1% BSA and incubated with Alexa Fluor 488 donkey anti‐mouse secondary antibody (A21202; Life Technologies, Paisley, UK) diluted to 1 µg/ml for 30 min at room temperature. After extensive washing (at least three times in PBS), cells were subjected to FACS sorting using either a DxP8 (Cytek, Ely, UK) or a MoFlo MLS cell sorter (DakoCytomation, London, UK).
3
Quantifying Retinal Ganglion Cell Survival
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All animals were euthanized seven days after I/R injury by CO2 inhalation followed by cervical dislocation. Eyes were enucleated and fixed in 4% paraformaldehyde for 2 hrs. As previously described47 (link)48 (link), retinas were immunostained for γ-synuclein, a marker for retinal ganglion cells (RGC), and the number of surviving RGC was determined. Briefly, retinas where incubated overnight with mouse anti-γ-synuclein primary antibody solution (1:400, Abnova Corporation, Walnut, CA, USA), followed by several rinses in PBS and incubation with an Alexa Fluor 488 donkey anti-mouse secondary antibody (1:300, Life technologies, Grand Island, NY). After another PBS wash, retinas were whole-mounted, cover slipped and imaged. Twelve images (318 × 318 μm, 40X magnifications) were taken at predetermined mid-peripheral locations using a Nikon Eclipse i80 confocal microscope (Nikon Instruments Inc, Melville, NY). γ-synuclein positive RGC were counted in a masked fashion by an independent observer using the cell counter plugin in ImageJ software (NIH).
4
Visualizing ABCA1 Localization in Macrophages
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For ABCA1 localization experiments, thioglycollate-elicited peritoneal macrophages were plated on coverslips and incubated with Ac-LDL (120 μg ml−1) for 24 h. At the end of incubation, cells were washed with PBS and incubated with fluorescently labelled cholera toxin B (CTxB) in 0.1% BSA in PBS at 4 °C for 20 min. Cells were then fixed with 4% PFA on ice for 10 min, permeabilized with 100% pre-chilled methanol at −20 °C for 2 min, blocked with 4% goat serum in PBS for 1 h and stained with monoclonal antibody against ABCA1 (1:1000; Abcam; clone AB.H10; # ab18180) overnight. After three washes with 1 × PBS, cells were incubated with Alexa Fluor 488 donkey anti-mouse secondary antibody (1:250, life technologies; # A21202) for 1 h at RT and coverslips were mounted on glass slides with mounting media with DAPI (Vectashield, Vector Laboratories; # H-1200). All images were analysed using a confocal microscope (Leica SP5 II) equipped with a 63X Plan Apo Lenses. All gains for the acquisition of comparable images were maintained at a constant level. Analysis of different images was performed using ImageJ (NIH) and Adobe Photoshop CS5.
5
Mitotic Cell Identification and Analysis
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Cells were harvested by trypsinization or by shake-off in cases that involved the collection of only mitotic cells. After being washed once with PBS, samples were fixed with cold 70% ethanol. Fixed cells were washed with PBS, permeabilized with 0.25% Triton X-100 (Sigma) in PBS and incubated with the MPM2 antibody (Millipore) diluted in PBS containing 2% BSA. After being washed with PBS containing 2% BSA, cells were incubated with Alexa Fluor 488 donkey anti-mouse secondary antibody (Life Technologies) diluted in PBS containing 2% BSA. After the antibody incubation, cells were washed again with PBS and resuspended in PBS containing 200 μg ml−1 DNase-free RNase A (QIAGEN) and 2 μg ml−1 propidium iodide (Sigma). Samples were analysed with a FACSCalibur flow cytometer (BD Biosciences) and the data were processed with the FlowJo software (Tree Star).
6
HeLa and MEF Cells for m6A Research
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HeLa (cervical cancer) was originally purchased from ATCC and MEF cells were a gift from D.J. Kwiatkowski (Harvard Medical School). Cells were not authenticated recently but tested negative for mycoplasma contamination. Both cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS). Antibodies used in the experiments are listed below: anti-YTHDF2 (Proteintech 24744-1-AP, 1:1000 WB, 1:600 IF); anti-Hsp70 (Stressgen SPA-810, 1:1000 WB); anti-FTO (Phosphosolutions 597-Fto, 1:1000 WB, 1:600 IF); anti-METTL3 (Abnova H00056339-B01P, 1:1000 WB, 1:600 IF); anti-METTL14 (sigma HPA038002, 1:1000 WB, 1:600 IF); anti-WTAP (Santa Cruz sc-374280, 1:1000 WB, 1:600 IF); anti-m6A (Millipore ABE572, 1:1000 m6A immunoblotting); Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen A21202. 1:600 IF); Alexa Fluor 546 donkey anti-rabbit secondary antibody (Invitrogen A10040, 1:600 IF).
7
Immunofluorescent Localization of CIB2 in Mouse Basilar Membrane
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All steps were performed at room temperature unless otherwise indicated. Mouse basilar membrane was dissected and fixed with cold 4% paraformaldehyde (PFA) in PBS for 30 min, then permeabilized and blocked with PBT1 (0.1% Triton X-100, 1% BSA and 5% heat-inactivated goat serum in PBS, pH 7.3) for 30 min. Samples were incubated overnight at 4°C with mouse anti-CIB2 polyclonal antibody (Abnova, Cat. No. H00010518-A01, 1:100 diluted) in PBT1 followed by incubation with Alexa Fluor® 488 donkey anti-mouse secondary antibody (Invitrogen, Cat. No. A21202, 1:300 diluted) in PBT2 (0.1% Triton X-100 and 0.1% BSA in PBS) for 1 h. After that, samples were incubated with TRITC-conjugated phalloidin (Sigma-Aldrich, Cat. No. P1951) in PBS for 30 min, then mounted in PBS/glycerol (1:1) and imaged with a confocal microscope (LSM 700, Zeiss, Germany) and a 40-fold objective. Immunostaining was performed at least three times for each genotype and age of mice tested.
8
Quantifying DNA Damage Response
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HeLa Tet-On cells transfected with the appropriate siRNAs were irradiated with 10 J/m2 UV. Samples were taken at the indicated timepoints, fixed with 70% ice-cold ethanol, blocked with PBS containing 5% BSA and 0.25% Triton-X100, and stained with the anti-γ-H2AX monoclonal antibody. Cells were washed, incubated with the Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen), and counterstained with propidium iodide in PBS containing RNase A. Cells were analyzed with a BD FACSCalibur flow cytometer by using the CellQuest software. Data were processed with FlowJo (FloJo, Ashland, OR).
9
HUVEC Angiogenesis Assay with Dll4 Expression
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HMVECs were transduced by the indicated adenovirus vector, as described previously, in basal growth medium for 12 h. Next, multiple wounds were made across the surface of each plate using the CellComb scratch assay system (EMD-Millipore). Cells were then treated with EBM-2MV, 0.4% FBS, and 20 ng/ml of VEGF or vehicle for 12 h. Following VEGF treatment, cells were harvested, washed with PBS, and incubated overnight at 4 °C with an anti-Dll4 mouse monoclonal antibody or mouse anti-IgG2b isotype control (Abcam, Cambridge, MA). The following day, cells were washed with PBS and incubated with an Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen), and the green fluorescence cell population (events = 3 × 10,000/well) was acquired as described previously (21 (link)) using the Guava EasyCyte system (Millipore) Data were analyzed and compared with the isotype control antibody using FlowJo software (Treestar).
10
HeLa and MEF Cells for m6A Research
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