Autoquant x software

Manufactured by Media Cybernetics

Autoquant X software is a digital imaging and analysis tool designed for microscopy applications. It provides functionality for image acquisition, processing, and analysis. The software is capable of performing quantitative measurements and analysis on various types of microscopy data.

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Lab products found in correlation

Orca r2, by Hamamatsu Photonics (4 mentions) Alexa fluor 488, by Thermo Fisher Scientific (4 mentions) Alexa fluor 568, by Thermo Fisher Scientific (4 mentions) Lsm 880 confocal microscope, by Zeiss (4 mentions) Zen software, by Zeiss (3 mentions) Axio imager z1, by Zeiss (3 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) Lsm800 confocal microscope, by Zeiss (2 mentions) Mouse anti α tubulin, by Merck Group (1 mentions) Rabbit anti β tubulin, by Abcam (1 mentions) Phosphate buffered saline (pbs), by Merck Group (1 mentions) Alexa fluor 647, by Thermo Fisher Scientific (1 mentions) Illustrator cs5, by Adobe (1 mentions) Dapi solution, by Merck Group (1 mentions) Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions) Zen blue, by Zeiss (1 mentions) Power block, by BioGenex (1 mentions) Amira software version 6, by Thermo Fisher Scientific (1 mentions) Vectashield mounting medium, by Vector Laboratories (1 mentions) Eclipse e600 microscope, by Nikon (1 mentions)

10 protocols using autoquant x software

Immunofluorescence Staining of Cellular Proteins

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For immunofluorescence processing, cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences) for 10 min followed by extraction with 0.3% Triton X-100 in PBS (Sigma-Aldrich) for 10 min. After blocking with 10% FBS in PBS with 0.1% Triton X-100, all primary antibodies were incubated at 4°C overnight. Then, the cells were washed with PBS containing 0.1% Triton X-100 and incubated with the respective secondary antibodies for 1 h at room temperature. Primary antibodies used were: mouse anti-Mad1 (1:500; Merck Millipore); mouse anti α-tubulin (1:2000; Sigma); rabbit anti-β-tubulin (1:2000; Abcam); anti-guinea pig CENP-C (1:1000; MBL International). Secondary antibodies used were Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 647 (1:1000; Themofisher). DNA was counterstained with 1 μg/mL DAPI (4',6'-diamino-2-fenil-indol; Sigma-Aldrich) and mounted onto glass slides with 20 mM Tris pH8, 0.5 N-propyl gallate and 90% glycerol. Images were acquired using an AxioImager Z1 (63x, Plan oil differential interference contract objective lens, 1.46 NA; from Carl Zeiss), coupled with a CCD camera (ORCA-R2; Hamamatsu Photonics) and the Zen software (Carl Zeiss). Blind deconvolution of 3D image datasets was performed using Autoquant X software (Media Cybernetics).

Gomes A.M., Orr B., Novais-Cruz M., De Sousa F., Macário-Monteiro J., Lemos C., Ferrás C, & Maiato H. (2022). Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells. Current Biology, 32(19), 4240-4254.e5.

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3D Widefield Imaging and Deconvolution

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3D wide-field image acquisition (0.23 μm z-step) was performed on an AxioImager Z1 (60× Plan-Apochromatic oil differential interference contrast objective lens, 1.46 NA, Carl Zeiss Microimaging Inc.) equipped with a CCD camera (ORCA-R2, Hamamatsu) operated by Zen software (Carl Zeiss, Inc.). Blind deconvolution of 3D image datasets was performed using AutoquantX software (Media Cybernetics). All images show maximum intensity projections. Adobe Photoshop 2021 and Adobe Illustrator CS5 (Adobe Systems) were used for histogram adjustments and panel assembly for publication.

Almeida A.C., Soares-de-Oliveira J., Drpic D., Cheeseman L.P., Damas J., Lewin H.A., Larkin D.M., Aguiar P., Pereira A.J, & Maiato H. (2022). Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals. Cell Reports, 39(1), 110610.

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Immunofluorescence Microscopy of Fixed and Live Cells

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Cells were cultured on glass coverslips (for fixed-cell) or on glass bottom dishes (for live-cell) in Complete Medium. For fixed-cell microscopy, cells were fixed with ice-cold 100% Methanol for 10 min and blocked with 10% Power Block (HK0855K; Biogenex Laboratories). Coverslips were incubated with primary antibody overnight at 4 °C followed by incubation with fluorescence-conjugated secondary antibody for 1 h at room temperature. Coverslips were counterstained with 1 μg/mL DAPI solution (D9542; Sigma) for 5 min and rinsed with PBS-T/PBS for 5 min. Then coverslips were mounted on slides with Prolong Glass Antifade Mountant (P36982; ThermoFisher, no DAPI). Slides were imaged with Zeiss LSM 880 confocal microscope. Live-cell microscopy were imaged with Andor spinning-disc confocal microscope with environmental CO₂ and temperature controls. Image deconvolution was conducted using AutoQuant X software (Media Cybernetics), then analyzed using Fiji and Zen Blue (Zeiss).

Tu X., Chu T.T., Jeltema D., Abbott K., Yang K., Xing C., Han J., Dobbs N, & Yan N. (2022). Interruption of post-Golgi STING trafficking activates tonic interferon signaling. Nature Communications, 13, 6977.

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3D Visualization of Nerve Regeneration

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Images were deconvolved using AutoQuant X Software (Media Cybernetics). Deconvolved images were then analysed using Amira Software version 6.0.1 (Thermo Scientific). Z-stacks were uploaded to create a 3D volume rendering of the crush site and labelled neurites. For samples containing more than one Z-stack, image stacks were aligned and merged using Amira’s merge module. The images were then rotated to position the crush site proximal to the retina on the left with regenerated neurites growing away from the crush site towards the right. The transformed data were resampled and axes swapped such that the XY plane became the cross-section along the length of the nerve. Images were cropped if needed to exclude any partial cross sections of the ends of the nerve caused by image rotation. Labelled regenerated neurites were segmented by thresholding pixels using Amira’s Segmentation Editor. The volume ratio of segmented neurites over total nerve volume was measured for each sample. Study results were then normalized and plotted as a bar graph. The ratio of segmented area over total nerve area for each cross-section was normalized and plotted as area ratio per slice. Area ratio plots were created starting from the behind the crush site proximal to the retina and following along the length of the nerve for the segmented regenerated neurites.

Siddiq M.M., Hannila S.S., Zorina Y., Nikulina E., Rabinovich V., Hou J., Huq R., Richman E.L., Tolentino R.E., Hansen J., Velenosi A., Kwon B.K., Tsirka S.E., Maze I., Sebra R., Beaumont K.G., Toro C.A., Cardozo C.P., Iyengar R, & Filbin M.T. (2021). Extracellular histones, a new class of inhibitory molecules of CNS axonal regeneration. Brain Communications, 3(4), fcab271.

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Immunofluorescence Imaging of Cultured Cells

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Cells were cultured on glass coverslips (for fixed-cell microscopy) or on glass-bottomed dishes (for live-cell microscopy) in complete medium. For fixed-cell microscopy, cells were fixed with 4% paraformaldehyde or 100% methanol (−20 °C) for 10 min, permeabilized with 0.2% Triton X-100 in PBS buffer and blocked with 3% BSA in PBS buffer. Coverslips were incubated with primary antibody for 1 h at room temperature followed by fluorescence-conjugated secondary antibody for 1 h at room temperature, then mounted on slides with VECTASHIELD mounting medium containing DAPI (Vector Laboratories). Slides were imaged with a Zeiss LSM 880 confocal microscope. Live-cell microscopy was imaged with an Andor spinning-disc confocal microscope with environmental CO₂ and temperature controls. Image deconvolution was conducted using AutoQuant X software (Media Cybernetics), then analysed using Image J. Co-localization was analysed by the Image J plug-in EzColocalization.

Chu T.T., Tu X., Yang K., Wu J., Repa J.J, & Yan N. (2021). Tonic prime-boost of STING signalling mediates Niemann–Pick disease type C. Nature, 596(7873), 570-575.

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Fluorescence and DIC Imaging of Live Cells

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Fluorescence and differential interference contrast (DIC) microscopy were performed using an Eclipse E600 microscope (Nikon, Tokyo, Japan). Cells expressing GFP or RFP fusion constructs were grown to mid–log phase in synthetic minimal medium and live imaged through fluorescein isothiocyanate or rhodamine filters. A series of z-stacks (1.6–3 μm in total depth with a 0.2-μm step for each section) were imaged by IPLab (Scanalytics, Milwaukee, WI). Raw data were deconvolved and analyzed by AutoQuant X software (Media Cybernetics, Rockville, MD). Fluorescence images shown are maximum projections of three-dimensional images.

Wang M, & Collins R.N. (2014). A lysine deacetylase Hos3 is targeted to the bud neck and involved in the spindle position checkpoint. Molecular Biology of the Cell, 25(18), 2720-2734.

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Sarcomere Localization of Exogenous cTnI

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Paraformaldehye-fixed, detergent-permeabilized myocytes were dual immunostained with the TnI Ab (MAB1691) plus fluorescein isothiocyanate (FITC)-conjugated secondary Ab pair followed by the FLAG Ab (Sigma) plus Texas Red (TR)-conjugated secondary Ab (TR) pair, 4 days after gene transfer, as described earlier (24 (link)). To establish sarcomere localization of exogenous cTnI, immunostained myocytes were analyzed using a Fluoview 500 laser scanning confocal microscope (Olympus; Center Valley, PA) for confocal projection imaging and de-convolution with Autoquant X software (Media Cybernetics; Rockville, MD).

Lang S.E., Stevenson T.K., Schatz T.M., Biesiadecki B.J, & Westfall M.V. (2017). FUNCTIONAL COMMUNICATION BETWEEN PKC-TARGETED CARDIAC TROPONIN I PHOSPHORYLATION SITES. Archives of biochemistry and biophysics, 627, 1-9.

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Super-Resolution Imaging Techniques for 3D

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3D wide-field images were acquired using an AxioImager Z1 (100× Plan-Apochromatic oil differential interference contrast objective lens, 1.46 NA, Carl Zeiss Microimaging Inc.) equipped with a CCD camera (ORCA-R2, Hamamatsu) operated by Zen software (Carl Zeiss, Inc.). Blind deconvolution of 3D image datasets was performed using Autoquant X software (Media Cybernetics). Confocal and super-resolution CH-STED images were acquired using Abberior Instruments “Expert Line” gated-STED coupled to a Nikon Ti microscope. An oil-immersion 60× 1.4 NA Plan-Apo objective (Nikon, Lambda Series) and pinhole size of 0.8 Airy units were used in all confocal acquisitions. Super-resolution images were acquired using a CH-STED beam (Pereira et al., 2019 (link)). A second LSM800 confocal microscope (Carl Zeiss Microimaging Inc.) mounted on a Zeiss-Axio imager Z1 equipped with plan-apochromat 63×/1.40 oil differential interference contrast (DIC) M27 objective (Carl Zeiss, Inc.) was used and operated by Zen 2008 software (Carl Zeiss, Inc.).

Ferreira L.T., Orr B., Rajendraprasad G., Pereira A.J., Lemos C., Lima J.T., Guasch Boldú C., Ferreira J.G., Barisic M, & Maiato H. (2020). α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity. The Journal of Cell Biology, 219(4), e201910064.

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Immunofluorescence of Trachea and Neutrophils

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For immunofluorescence microscopy of trachea, 10-µm thickness frozen trachea sections were prepared and stained with the indicated antibodies. Superblock (Thermo Scientific) was used as a blocker. Quantitative analysis of fluorescent signals was performed using NIS (Nikon) or Autoquant X software (MediaCybernetics). For immunofluorescence microscopy of neutrophils, cells were fixed with 2% paraformaldehyde and stained with the indicated antibodies in the presence of 0.025% saponin.

Lim K., Hyun Y.M., Lambert-Emo K., Capece T., Bae S., Miller R., Topham D.J, & Kim M. (2015). Neutrophil trails guide influenza-specific CD8+ T cells in the airways. Science (New York, N.Y.), 349(6252), aaa4352.

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Immunofluorescence Processing of Mitotic Proteins

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For immunofluorescence processing, cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences) for 10 min followed by extraction with 2 × 5 min washes using 0.5% Triton X-100 (Sigma-Aldrich). Primary antibodies used were: anti-mouse Aurora B (Monoclonal AIM-1; BD Biosciences; 1:250); anti-mouse INCENP (Monoclonal; Santa Cruz Biotechnology; 1:50); anti-rabbit Mklp2/Kif20A (Bethyl Laboratories; 1:2000); anti-rabbit Phospho-Histone H3 Ser10 (Monoclonal; D2C8; Cell Signaling Technology; 1:5000), anti-rabbit Kif4a (Thermofisher; 1:1000); human anti-centromere antibodies (1:2000; Fitzgerald); mouse anti-pT210 PLK1 (1:1000; Abcam). Secondary antibodies used were Alexa Fluor 488 (Themofisher; 1:2000) and Alexa Fluor 568 (Themofisher; 1:2000). DNA was counterstained with 1 μg/mL DAPI (4’,6’-diamino-2-fenil-indol; Sigma-Aldrich) and mounted onto glass slides with 20 mM Tris pH8, 0.5 N-propyl gallate and 90% glycerol. 3D wide-field images were acquired using an AxioImager Z1 (100x Plan-Apochromatic oil differential interference contrast objective lens, 1.46 NA, ZEISS) equipped with a CCD camera (ORCA-R2, Hamamatsu) operated by Zen software (ZEISS). Blind deconvolution of 3D image datasets was performed using Autoquant X software (Media Cybernetics).

Orr B., De Sousa F., Gomes A.M., Afonso O., Ferreira L.T., Figueiredo A.C, & Maiato H. (2021). An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors. Cell Reports, 37(6), 109783.

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