passages.id: PMC-1072800-sec-07__text
passages.type: sec
passages.text: Immunostaining and confocal analysis
  DNA labelling and staining with 5-bromo-2'-deoxyuridine (BrdU labelling and detection kit I; Boehringer Mannheim, Germany) was performed according to the manufacturer's instructions. The nucleoli were detected with an anti-nucleolin antibody (clone 3G4B2; Upstate biotechnology, Lake Placid, NY) on cells fixed in 2% formalin for 10 min followed by permeabilization with ice-cold methanol for 30 min. The secondary antibody used for both BrdU and nucleoli staining was a rhodamine (tetra-methyl)-conjugated goat anti-mouse antibody (T-2762) from Molecular Probe (Eugene, OR). The cells were examined in a Zeiss LSM 510 laser scanning microscope equipped with a Plan-Apochromate 63x/1.4 oil immersion objective. We used the 488 nm laser line for excitation of EGFP (detected at 505 nm < lambdaEGFP < 530 nm) and the 543 nm laser line for rhodamine (tetra-methyl) (detected at lambdaRhodamine > 560 nm). ECFP fusion protein was excited with a 458 nm laser line (detected at 480 nm < lambdaECFP < 520 nm), EYFP fusion protein was excited with a 514 nm laser line (lambdaEGFP > 560 nm) and HcRed fusion protein was excited with a 543 nm laser line and detected at >585 or 650 nm. The images were from 1 mum thick slices of the cells. The images were exported into Adobe Photoshop (Adobe Systems Inc., San Jose, CA).
  