Computational protocol: Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

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Protocol publication

[…] Proteins were extracted in RIPA buffer and concentrations were measured by the bicinchoninic acid method (Pierce, USA). Total extracts were electrophoretically fractionated on 10 or 12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to a 0.45 μm nitrocellulose membrane. Membranes were blocked for 1 h with 5% nonfat dry milk in TBS-Tween and incubated overnight at 4°C with the following primary antibodies: pH2AX, p27, pAkt, pErk1/2 (all of them from Cell Signaling, diluted 1:1000), p21 (Dako, 1:1000), telomerase (Novocastra, 1:1000), dyskerin (Sigma, 1:1000), and β-actin (Sigma, 1:5000). Membranes were washed and incubated with peroxidase-labeled secondary antibodies at room temperature for 1 h. Immunoreactive bands were detected with the Lumi-Light Western Blotting Kit (Roche, Switzerland) and quantified with ImageJ (NIH, USA). [...] For quantification of immunohistochemistry, 60 random images (x200) per experimental group were captured with a Nikon microscope Y-TSH (Japan). The stained areas were quantified with the ImageJ software and data were expressed as positive immunostained area with respect to reference area.For quantification of immunofluorescence, thirty plane image stacks were captured 0.29 μm apart. Images were acquired with a motorized Axioplan 2ie (Zeiss, Germany) epifluorescence microscope Plan Apochromat. Images were recorded using a CCD Photometrics monochrome camera (Photometrics® CoolSNAP™cf, Microimaging Application Group, USA). The Metamorph© software (Molecular Devices, Canada) was used to control the image acquisition.Deconvolution, linear unmixing and image segmentation were applied following previously published protocols in order to reduce the background signal and improve quantification accuracy []. Briefly, software-based image deconvolution was applied to obtain 3D quasi-confocal sectioning from the wide field image stacks. The model of the Point-Spread Function (PSF) was experimentally calculated for each channel using the TetraSpeck™ Fluorescent Microspheres (Molecular Probes, USA) kit. Deconvolution was performed with the Huygens software (Scientific Volume Imaging BV, The Netherlands). Cross-talk between image channels was estimated and eliminated using a Non Negative Least Squares (NNLS) algorithm [].Telomere length was estimated as previously described []. A total of 100 nuclei per mice (n = 4) were analyzed. Data are expressed as average of telomeric length. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Diseases Carcinoma, Non-Small-Cell Lung, Lung Neoplasms, Neoplasms, Brain Stem Neoplasms