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Mouse monoclonal anti cytc antibody

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The Mouse monoclonal anti-CytC antibody is a laboratory reagent used for the detection and analysis of the cytochrome C (CytC) protein. It is a specific antibody produced in mice that binds to the CytC antigen.

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Lab products found in correlation

Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (4 mentions) 12 mm poly d lysine coated glass coverslips, by BD (2 mentions) Mitrotracker red cm h2x ros, by Thermo Fisher Scientific (2 mentions) Lsm 510, by Zeiss (2 mentions) Glass chamber slide, by Thermo Fisher Scientific (1 mentions) Immobilon, by Merck Group (1 mentions) Hrp labeled anti mouse igg, by GE Healthcare (1 mentions) Hrp labeled anti rabbit igg, by GE Healthcare (1 mentions) Lsm 510 microscope, by Zeiss (1 mentions) Retinoic acid, by Merck Group (1 mentions) Optical borosilicate poly l lysine coated sterile glass covers, by Thermo Fisher Scientific (1 mentions)

5 protocols using mouse monoclonal anti cytc antibody

1

Immunocytochemical Analysis of Cytochrome C

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Both SH-SY5Y cells and Glioma cells were plated on glass chamber slides (Thermo Fisher Scientific, Rochester, NY). Slides were pre-coated with poly-D-lysine for SH-SY5Y cells. After seeding, cells were allowed to attach for 1 day prior to treatment with Aβ in presence or absence of MTZ for 24 hours. Cells were then washed with PBS, fixed with 4% paraformaldehyde (10 min, RT), washed again, and blocked for 1 hour with 20 mg/ml BSA in PBS containing 0.3% Triton X-100 (PBST). Slides were further incubated with mouse monoclonal anti-CytC antibody (BD Biosciences; 1:200 in PBST containing 5 mg/ml BSA; 2h, RT) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, Grand Island, NY) 1:200 in PBST with 5 mg/ml BSA for 1h at RT, as previously described (Fossati et al., 2013 ). Fluorescence signals were visualized in a Zeiss LSM 510 laser scanning confocal/Confocor2 microscope using a 40x DIC oil immersion objective and LSM 510 software; acquired images were processed and analyzed using ImageJ (National Institute of Health; http://rsbweb.nih.gov/ij/).
Fossati S., Giannoni P., Solesio M.E., Cocklin S.L., Cabrera E., Ghiso J, & Rostagno A. (2015). The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain. Neurobiology of disease, 86, 29-40.
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2

Western Blot Analysis of Cellular Fractions

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Cytosolic and mitochondrial fractions were separated on 14% SDS-polyacrylamide gels under reducing conditions and electrotransferred to PVDF membranes (Immobilon, Millipore, Billerica, MA; 0.45 μm pore; 400 mA, 1.5h) using CAPS buffer, as above. Membranes were blocked with 5% nonfat milk in TBST, and subsequently immunoreacted with mouse monoclonal anti-CytC antibody (BD; 1 μg/ml in 5 % nonfat milk in TBST, overnight, 4°C) followed by HRP-labeled anti-mouse IgG (1:10,000; GE Healthcare), and ECL detection, as above. As loading controls, membranes were probed with rabbit polyclonal anti-β actin (Novus Biologicals; 1 μg/ml, overnight, 4°C) followed by HRP-labeled anti-rabbit IgG (1: 5,000; GE Healthcare). Densitometric assessment of band intensities was performed using ImageJ software (rsbweb.nih.gov)
Fossati S., Giannoni P., Solesio M.E., Cocklin S.L., Cabrera E., Ghiso J, & Rostagno A. (2015). The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain. Neurobiology of disease, 86, 29-40.
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3

SH-SY5Y Cells Treated with ADan Peptides

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SH-SY5Y cells were seeded on 12 mm poly-D-lysine coated glass coverslips (BD Biosciences, Franklin Lakes, NJ) at a density of 7 × 104 cells/coverslip and allowed to attach for 1 day prior to 6 day differentiation in DMEM (Mediatech) with 10% FBS and 10 μM retinoic acid (Sigma). Cells were subsequently treated with 50 μM ADan pE or ADan E for up to 24h, fixed with 4% paraformaldehyde, and blocked with 20 mg/ml BSA in PBS containing 0.3% Triton X-100. After incubation with mouse monoclonal anti-cyt c antibody (BD Biosciences; 1:200 in PBS containing 5 mg/ml BSA, 2h at RT) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies; 1:200 in PBS with 5 mg/ml BSA, 1h at RT), nuclei were counterstained with TO-PRO-3 iodide (Life Technologies; 1:1,000) as previously described [36 (link)-38 (link)]. To examine cyt c mitochondrial localization in conjunction with changes in the membrane potential of the organelle, cells –after peptide treatment– were washed with warm PBS and incubated for 30 minutes with 1.5 μM Mitrotracker Red CM-H2X Ros (Life Technologies), followed by cyt c immunostaining as above. All images were acquired using a Zeiss LSM 510 microscope and analyzed using Image J.
Todd K., Ghiso J, & Rostagno A. (2015). Oxidative stress and mitochondria-mediated cell death mechanisms triggered by the familial Danish dementia ADan amyloid. Neurobiology of disease, 85, 130-143.
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4

Cytochrome c Localization in Peptide-Treated SH-SY5Y Cells

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SH-SY5Y cells were seeded on 12 mm poly-D-lysine coated glass coverslips (BD Biosciences, Franklin Lakes, NJ) at a density of 7 x 104 cells/coverslip and allowed to attach for 1 day prior to treatment with 50 μM ABri pE/E, Bri1-23 pE/E, or Aβ42 for 4–16 hours. After washing once with cold PBS, cells were fixed with 4% paraformaldehyde and blocked with 20 mg/ml BSA in PBS containing 0.3% Triton X-100. Cells were subsequently incubated with mouse monoclonal anti-cyt c antibody (BD Biosciences; 1:200 in PBS containing 5 mg/ml BSA, 2h at RT) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, Carlsbad, CA; 1:200 in PBS with 5 mg/ml BSA, 1h at RT), and nuclei were counterstained TO-PRO-3 iodide (Life Technologies; 1:1,000 in PBS, 10 minutes at RT) as previously described. To examine mitochondrial localization in conjunction with changes in the membrane potential of the organelle, cells–after peptide treatment–were washed once with warm PBS and incubated for 30 minutes with 1.5 μM Mitrotracker Red CM-H2X Ros (Life Technologies), followed by cyt c immunostaining as above. All images were acquired using a Zeiss LSM 510 confocal microscope and analyzed using Image J (NIH, Bethesda, MD; http://rsbweb.nih.gov).
Todd K., Fossati S., Ghiso J, & Rostagno A. (2014). Mitochondrial dysfunction induced by a post-translationally modified amyloid linked to a familial mutation in an alternative model of neurodegeneration. Biochimica et biophysica acta, 1842(12 0 0), 2457-2467.
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5

Cytochrome C Localization in Neuronal and ECs

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SH‐SY5Y and microvascular ECs cells were plated on 15‐mm optical borosilicate poly‐L‐lysine‐coated sterile glass covers (Thermo Fisher, Waltham, Massachusetts, USA) at a 70% confluence. After 24 hr, cells were treated with the peptides in the presence or absence of MTZ or ATZ for 16 hr. Cells were then washed with PBS, fixed with 4% paraformaldehyde (10 min, RT), washed again, and blocked with 20 mg/ml BSA in PBS containing 0.3% Triton X‐100 (PBST). Slides were further incubated with mouse monoclonal anti‐CytC antibody (BD Biosciences; 1:200 in PBST containing 5 mg/ml BSA; 2 hr, RT) followed by Alexa Fluor 488‐conjugated anti‐mouse IgG (Thermo Fisher, Waltham, Massachusetts, USA) 1:200 in PBST with 5 mg/ml BSA for 1 hr at RT, as previously described fluorescence signals were visualized in a Zeiss LSM 510 laser scanning confocal/Confocor2 microscope using a 40× DIC oil immersion objective and LSM 510 software; acquired images were processed and analyzed using ImageJ (National Institute of Health).
Solesio M.E., Peixoto P.M., Debure L., Madamba S.M., de Leon M.J., Wisniewski T., Pavlov E.V, & Fossati S. (2018). Carbonic anhydrase inhibition selectively prevents amyloid β neurovascular mitochondrial toxicity. Aging Cell, 17(4), e12787.
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