Anti mouse alexa fluor 488

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany

Anti-mouse Alexa Fluor 488 is a fluorescently-labeled secondary antibody that specifically binds to mouse primary antibodies. It is designed for use in various immunodetection techniques, such as Western blotting, immunohistochemistry, and flow cytometry.

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Alexa Fluor 594/488-conjugated goat anti-mouse/rabbit secondary antibody Alexa Fluor 488-conjugated anti-mouse IgG Alexa Fluor 488-conjugated goat anti-mouse IgM Alexa Fluor 488-conjugated anti-mouse IgG F(ab′)2 fragment Alexa Fluor 488-conjugated donkey anti-mouse antibody Alexa Fluor 488 donkey anti-mouse IgG antibody Alexa Fluor 488-AffiniPure F(ab’)2 Fragment goat anti-mouse IgG Alexa Fluor 488 goat anti-mouse or rabbit IgG Donkey anti-mouse conjugated to Alexa Fluor 488 Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies

195 protocols using anti mouse alexa fluor 488

Visualizing Lysosomal Targeting of Protein Therapeutics

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HeLa cells were incubated with LTM-mCherry (0.5 μM), mCherry-LTM (0.5 μM), or LTM-TPP1 (2 μM) for 2 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. mCherry constructs were visualized with a rabbit polyclonal antibody against mCherry (Abcam, ab16745) and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific). LAMP1 was stained with a mouse primary antibody (DSHB 1D4B) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific).
Colocalization was quantified using the Volocity (PerkinElmer) software package to measure Manders’ coefficients of mCherry signal with LAMP1 signal. The minimal threshold for the 488- and 568-nm channels was adjusted to correct the background signal. The same threshold for both channels was used for all the cells examined.
CLN2^−/− fibroblast 19494 were incubated with LTM-TPP1 (2 μM) for 2 hours. Cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. LTM-TPP1 was visualized with a mouse monoclonal against TPP1 (Abcam, ab54685) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific). LAMP1 was stained with rabbit anti-LAMP1 and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific).

Sugiman-Marangos S.N., Beilhartz G.L., Zhao X., Zhou D., Hua R., Kim P.K., Rini J.M., Minassian B.A, & Melnyk R.A. (2020). Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy. Science Advances, 6(50), eabb0385.

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Whole-cell and LFP Recordings in Hippocampus

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For LFP recordings, glass microelectrodes (tip diameter ~5–10 μm; resistance: 0.2–0.3 MΩ) were filled with ACSF before use. Whole-cell recordings were performed with borosilicate glass electrodes (2–5 MΩ) filled with (in mM) 120 K-gluconate, 10 HEPES, 10 KCl, 3 Mg-ATP, 5 EGTA, 2 MgSO₄, 0.3 Na-GTP and 14 phosphocreatine. The pH was adjusted to 7.4 with KOH. LFP signals in the CA3 pyramidal cell layer were amplified 1,000-fold, filtered (1–8 kHz), and sampled at 20 kHz. Whole-cell and extracellular recordings were performed using a Multiclamp 700A amplifier (Axon Instruments). For parallel double patch-clamp and field recordings, a custom-made two channel extracellular amplifier was used. Cells were routinely loaded with 0.2% biocytin. After recordings, slices were transferred to 4% paraformaldehyde. Biocytin-filled cells were subsequently visualized with streptavidin conjugated with Dy-Light 488. After acquisition of confocal images, neuronal reconstruction was performed with the imageJ package (Schneider et al., 2012 (link)). To better estimate the slice position along the dorso-ventral axis slices were either Nissl stained or stained with anti-NeuN (Millipore) or anti-calbindin (Swant) antibodies followed by the secondary polyclonal antibody anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 555 (TermoFisher), respectively.

Imbrosci B., Nitzan N., McKenzie S., Donoso J.R., Swaminathan A., Böhm C., Maier N, & Schmitz D. (2021). Subiculum as a generator of sharp wave-ripples in the rodent hippocampus. Cell reports, 35(3), 109021.

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Whole-cell and LFP Recordings in Hippocampus

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Analyzing AURKA-Venus Localization in U2OS Cells

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U2OS cells were transfected with AURKA-Venus WT or mutant and seeded onto coverslips. After 24 h, cells were fixed with ice-cold methanol (IF) or 4% PFA (isPLA). Cells were processed for IF using primary antibodies against GFP (rabbit polyclonal, ab290; Abcam) and β-tubulin (mouse mAb, T4026; Sigma-Aldrich) and secondary antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific). Cells were processed for isPLA using Duolink In Situ Detection Orange (Sigma-Aldrich) according to the manufacturer’s instructions, using primary antibodies against GFP (mouse mAb, clone #11814460001; Roche) and TPX2 (rabbit polyclonal antibody; Novus Biological). Epifluorescence images were acquired on the widefield imaging platform described above, as stacks of 500-nm step with 2 × 2 bin, using appropriate filter sets and 40× NA 1.3 oil objective, and exported as maximal intensity projections in ImageJ (http://rsb.info.nih.gov/ij/; National Institutes of Health).

Abdelbaki A., Ascanelli C., Okoye C.N., Akman H.B., Janson G., Min M., Marcozzi C., Hagting A., Grant R., De Luca M., Asteriti I.A., Guarguaglini G., Paiardini A, & Lindon C. (2022). Revisiting degron motifs in human AURKA required for its targeting by APC/CFZR1. Life Science Alliance, 6(2), e202201372.

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Immunohistochemical Profiling of Murine Brain Regions

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Coronal cryosections (16–20 μm) were collected serially in a cryostat and incubated at room temperature for 60 min with a blocking solution consisting of 2% BSA, 0.3% Triton X-100 in PBS. The following primary antibodies were diluted in the same solution and incubated overnight at 4°C: mouse anti-Goα (1:500; #MAB3073, Merck Millipore), mouse anti-Pcp2 (1:500; #sc-137064, Santa Cruz), mouse anti-Cacna1s (1:250; #MAB427, Merck Millipore), rabbit anti-recoverin (1:500; #AB5585, Merck Millipore), rabbit anti-iba1 (1:500; #234 013, Synaptic Systems), and rat anti-Cd11b (1:500; #101202, BioLegend). Following several washes in PBS, the sections were incubated for 2 h at room temperature with the following secondary antibodies, all diluted 1:500 in blocking solution: anti-mouse Alexa Fluor 488 (#A-21121), anti-rat Alexa Fluor 488 (#A-21208), anti-rabbit Alexa Fluor 488 (#A-11008), anti-mouse Alexa Fluor 555 (#A-21127), and anti-rabbit Alexa Fluor 647 (#A-32733, all antibodies from Thermo Fisher Scientific). The sections were then washed in PBS and counterstained with DAPI (#D1306, Thermo Fisher Scientific) prior to mounting (#18606-5, Aqua-Poly/Mount).

Murenu E., Pavlou M., Richter L., Rapti K., Just S., Cehajic-Kapetanovic J., Tafrishi N., Hayes A., Scholey R., Lucas R., Büning H., Grimm D, & Michalakis S. (2021). A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting. Molecular Therapy. Methods & Clinical Development, 20, 587-600.

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Immunostaining and Axon Length Analysis

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Hippocampal neurons (4 DIV) were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.02% saponin and blocked with 10 mM glycine and 5% BSA in TBS. Staining was performed with mouse anti-SMI312 (1:500; Abcam), chicken anti-GFP (1:1000; Abcam) and/or phalloidin-Alexa594 (1:40; ThermoFisher Scientific) and detected with anti-mouse AlexaFluor488 or 594 secondary antibodies (1:400; ThermoFisher Scientific) and Hoechst 33258. Images were captured with a Nikon i90 fluorescence microscope and analyzed with Image J (NIH, USA) and Metamorph (Molecular Devices, Sunnyvale, CA, USA) softwares. Axons were manually tracked by following the neurofilament staining from the cell body to the actin neurite tip or GFP staining. Axon length was obtained from at least three independent experiments counting three-six independent coverslips (n = 30 neurons/coverslip) per condition. For axonal length imaging using NMIIA mutants, images were captured with a Zeiss LSM700 laser scanning microscope and analyzed with FilamentTracer tool from Imaris 8.1 Software (Bitplane AG, Zurich, Switzerland).

Javier-Torrent M., Marco S., Rocandio D., Pons-Vizcarra M., Janes P.W., Lackmann M., Egea J, & Saura C.A. (2019). Presenilin/γ-secretase-dependent EphA3 processing mediates axon elongation through non-muscle myosin IIA. eLife, 8, e43646.

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Immunodetection of Ca2+ Channel Subunits

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Antibodies (Abs) used were: anti-Ca_V2.2 II-III loop Ab (rabbit polyclonal) (Raghib et al., 2001 (link)), anti-Ca_V2.1 II-III loop Ab (rabbit polyclonal, Alomone), anti- α₂δ-1 Ab (mouse monoclonal, Sigma-Aldrich), anti-HA (rat monoclonal, Roche), anti-HA (rabbit polyclonal, Sigma), anti-PDI (mouse monoclonal, Abcam), anti-Akt Ab (rabbit polyclonal, Cell Signaling Technology) and anti-GAPDH Ab (mouse monoclonal, Ambion). For immunocytochemistry, secondary Abs (1:500) used were anti-rabbit-Alexa Fluor 594, anti-rat- Alexa Fluor 488, anti-rat-Alexa Fluor 594 and 647 and anti-mouse-Alexa Fluor 488 (ThermoFisher). For immunoblotting, secondary Abs (1:2000) were anti-rabbit-Horseradish Peroxidase (HRP), and anti-mouse HRP (Biorad).

Meyer J.O., Dahimene S., Page K.M., Ferron L., Kadurin I., Ellaway J.I., Zhao P., Patel T., Rothwell S.W., Lin P., Pratt W.S, & Dolphin A.C. (2019). Disruption of the Key Ca2+ Binding Site in the Selectivity Filter of Neuronal Voltage-Gated Calcium Channels Inhibits Channel Trafficking. Cell Reports, 29(1), 22-33.e5.

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Mitochondrial and Prohibitin Labeling

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1×10⁴ Cells were plated onto a 24 well plate over a 30 mm coverslip. Mitotracker Red CMXRos (Life Technologies®) was used to label the mitochondria. Mitotracker Red was diluted in each cell culture medium for 20 min at a concentration of 500 nM and incubated at 37°C in the presence of 5% CO₂. Cells were washed with PBS and then fixed in 4% paraformaldehyde in PBS for 15 min. After three washes with PBS, 0.2% Triton X-100 in PBS was added for 5 min for cell permeabilization. Nonspecific sites were blocked with PBS with 5% BSA for 1 h. Mouse monoclonal antibody to PHB MS-261-P0 (Thermofisher®) (4 μg/mL), was incubated overnight at 4°C in PBS with 5% BSA. Secondary antibody conjugated with anti-mouse Alexa Fluor 488 (Thermofisher®) (4 μg/mL) and the nuclear marker Hoechst 33258® was incubated for 1 h at room temperature in PBS containing 5% BSA. The analysis was performed using a Zeiss LSM 510® Meta/ UV confocal microscope.

Tortelli TC J.u.n.i.o.r., de Godoy L.M., de Souza G.A., Bonatto D., Otake A.H., de Freitas Saito R., Rosa J.C., Greene L.J, & Chammas R. (2017). Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death. Oncotarget, 8(26), 43114-43129.

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Immunofluorescence Assay for Leishmania Parasites

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Promastigotes from the two strains were centrifuged, washed with PBS, and fixed with 4% paraformaldehyde for 30 min. Parasites were washed in PBS, transferred to glass slides, and air dried. Slides were incubated for 30 min in 50 mM ammonium chloride and for 10 min with TBS containing 1% BSA and 0,1% Triton, and then blocked for 30 min with PBS containing 1% de BSA at 37 °C. Cells were then incubated with anti-LPG (Thermo Fisher Scientific, Waltham, MA, USA) 1:1000 overnight at 4 °C. Slides were washed with PBS and incubated with DAPI (10 mg/mL) and anti-mouse Alexa fluor 488 (Thermo Fisher Scientific, USA) for 1 h at 4 °C. Slides were washed 10 times in PBS, 3 times in water, and fixed with ProLong^® (Life Technologies, Carlsbad, CA, USA).

Tano F.T., Telleria E.L., Rêgo F.D., Coelho F.S., de Rezende E., Soares R.P., Traub-Cseko Y.M, & Stolf B.S. (2023). Phenotypical Differences between Leishmania (Leishmania) amazonensis PH8 and LV79 Strains May Impact Survival in Mammal Host and in Phlebotomine Sand Flies. Pathogens, 12(2), 173.

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Immunofluorescent Analysis of DNA Damage

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We performed immunofluorescent cell microscopy studies to analyze DNA damage measured by γH2AX foci formation. OVCAR8 cells were plated on cover slips in 24 well-plates 24 hrs prior to treatment with 0.01, 0.1, 1, or 10 μM olaparib, 10e, 15b, or DMSO controls. Treatment with PARPi lasted for 24 hrs and at that time media and drug was removed, cells were fixed with 500 μL/well of 4% paraformaldehyde in PBS for 10 minutes. Following fixation cells were washed three times with PBS then permeabilized using 500μL/well triton-x. Cover slips with cells were then removed from the 24-well plate and were placed on a flat parafilmed surface with the cells exposed to air. Primary for antibody anti-phospho-histoneH2A.X (EMD Millipore) was diluted 1:5000 in a 0.2% tween 20 PBS (0.2% PBST) solution and 30 μL was added to cover slips. Primary antibody was incubated at 37 °C for 1 hr at which time they were returned to the 24-well plate and washed 3 times with 0.2% PBST. Next secondary antibody (anti-mouse alexafluor488, thermofisher) was diluted 1:100 in 0.2% PBST and added following the same steps and incubation time used for the primary antibody. Next, cover slips were returned to 24-well plate and washed with 0.2% PBST 3 times, removed from plate, and mounted on slides using DAPI mounting medium.

Reilly S.W., Puentes L.N., Wilson K., Hsieh C.J., Weng C.C., Makvandi M, & Mach R.H. (2018). Examination of Diazaspiro Cores as Piperazine Bioisosteres in the Olaparib Framework Shows Reduced DNA Damage and Cytotoxicity. Journal of medicinal chemistry, 61(12), 5367-5379.

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