Similar protocols

Protocol publication

[…] Cells and tissues were visualized using a Leica microscope (model DMR, Melville NY) fitted with 40X (PL Fluotar, NA 1.0) and 63X (PL APO, NA 1.32) objectives. Images on the DMR microscope were acquired using an Orca 100 CCD camera (model C4742-95; Hamamatsu, Bridgewater, NJ) and analyzed using ImageJ software (NIH version 2.0.0) or Metamorph. For N-SIM analysis, the samples were illuminated with spatially high-frequency patterned excitation light (100X objective lens, NA 1.49; TiE N-SIM microscope [Nikon] and iXON X3 897 camera [Andor Technology]). Images were reconstructed and analytically processed to reconstruct subresolution structure of the samples using Elements version 4 software (Nikon). For siNEDD8 and siCOPS3 EGFR internalization, the Axioimager and the Apotome2 was used with the 40x objective and a MIP of the Z sections was used. [...] All experiments were performed independently >3 times (i.e. biological replicates performed on different days, not technical replicates performed in parallel at the same time). For each independent/biological replicate, multiple experimental/control arms were processed and analyzed in parallel. Representative experiments are displayed throughout the figures and where indicated in the figure legends, quantification of experiments is reported as mean ±standard error mean (SEM). For biochemical analyses, cell lysates were derived from a population of cells grown in 10 cm dishes (approximately 4 million cells) or 3 cm dishes (approximately 1 million cells). For immunofluorescence or PLA analyses, cells were plated onto a coverslip in a 3 cm dish and quantified as follows: PLA particles were counted for 5–10 randomly selected fields, each containing 20–100 cells (depending on whether image was obtained using a 40X or 63X objective and/or whether the image was from cultured cells or 3D organotypic rafts). Parallel analyses of protein specific antibodies with IgG (mouse or rabbit) were performed to control for false positives. Fluorescence pixel intensity at randomly selected cell borders was determined by multiplying the mean pixel intensity by the area of the defined border divided by the border length. Background intensity was randomly selected from an area on the image and subtracted from the border intensity. Two group comparisons were performed using two-tailed, two-sample equal variance Student’s t test using Excel (Microsoft, Redmond, WA, USA). p-values<0.05 were considered statistically significant. Densitometric analyses were performed on scanned films of immunoblots with the lightest possible exposure, and numbers derived from the densitometric analyses were displayed below immunoblots as indicated in the figures. All densitometric quantifications were normalized to a protein standard, as indicated in figures. All immunofluorescence and densitometric calculations were performed using FIJI (FIJI Is Just ImageJ) (), ImageJ () or Metamorph. […]

Pipeline specifications

Software tools ImageJ, MetaMorph, Fiji
Applications Super-resolution imaging, Microscopic phenotype analysis
Diseases Carcinoma, Neoplasms