Computational protocol: The Differential Formation of the LINC-Mediated Perinuclear Actin Cap in Pluripotent and Somatic Cells

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Protocol publication

[…] Actin filament and focal adhesion architecture were examined by immunofluorescence brightfield and confocal microscopy. Samples were fixed with 3.7% paraformaldehyde for 1 h, and stained for nuclear DNA, filamentous actin, tumor recognition antigen 1–81 (TRA-1-81), and nuclear envelope proteins lamin A/C, Nesprin2 giant, Nesprin3, and Sun2. For staining, cells were permeabilized with 0.1% Triton X-100 for 10 min. Goat serum, 10%, in phosphate-buffered saline was used to block nonspecific binding for 20 min. The primary antibodies used were: anti-TRA-1-81 antibody (Millipore MAB4381, Billerica, MA) at 1∶100; anti-lamin A/C (Abcam AB26300, Cambridge, MA) at 1∶500; anti-Sun2 (provided by Dr. Didier Hodzic, Washington University School of Medicine, at St. Louis) and anti-Nesprin 3 (provided by Dr. A. Sonnenberg, The Netherlands Cancer Institute, Amsterdam, The Netherlands) at 1∶2000 and 1∶1000, respectively; and anti-Nesprin 2 giant (provided by Drs. E. Gomes and G.G. Gundersen, Columbia University, New York) at 1∶500. Secondary treatments were done with Alexa-Fluor goat-anti-rabbit 488 or 568. Both primary and secondary antibody treatments were conducted for 1 h. To visualize actin filaments and nuclear DNA, Alexa-Fluor phalloidin 488 or 568 and 300 nM DAPI (Invitrogen, Carlsbad, CA) were used, respectively.Fluorescent images were either collected using a Cascade 1 K CCD camera (Roper Scientific, Tucson, AZ) mounted on a Nikon TE2000E microscope with a 60× Plan Fluor objective (N.A. 1.4) or using a Zeiss 510 laser-scanning confocal microscope with a 63× Plan-Apochromat objective (N.A. 1.4). Three-dimensional images were analyzed and processed using a combination of Zeiss LSM Image Browser (Zeiss), Metaporph, and ImageJ (NIH). Special attention was paid to use small increments between focal sections (<0.3 µm) and to scan the same cell starting at slightly different heights as to not miss actin structures underneath the nucleus.DAPI-stained nuclei were individually traced by hand and size, length of minor axis, length of major axis, and shape factor were measured using Metamorph software (Universal Imaging, Downingtown, PA). Mean values, standard error of measurement (SEM), and statistical analysis were calculated and plotted using Graphpad Prism (Graphpad Software, San Diego, CA). Two-tailed unpaired t tests were conducted to determine significance. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens