Donkey anti mouse conjugated with tritc

For immunofluorescence experiments, cells were plated on glass coverslips, fixed in 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were incubated in primary antibody solution overnight at 4°C, washed with PBS, and incubated for 1 hour with secondary antibody solution at room temperature and washed again in PBS. Coverslips were mounted on glass slides using DABCO mounting medium (Fluka) with DAPI. Staining was visualized on a Zeiss 510 Meta confocal microscope using the 63X objective. Primary antibodies used for staining were mouse anti-human maspin (BD Pharmingen, 1:25), mouse anti-human Ki-67 (DakoCytomation, 1:50), rabbit anti-human lamin C (a kind gift from prof. C.J Hutchison, 1:20). Secondary antibodies were donkey anti-mouse conjugated with TRITC and donkey anti-rabbit conjugated with Cy5 (both from Jackson ImmunoResearch). After 24 hours to 120 hours following transfection, EGFP-expressing cells were counted for expression of Ki-67 antigen in five to ten fields of view and the Ki-67-positive subpopulation was calculated as the percentage of all transfected cells.

Cells were plated on glass coverslips, fixed in 4 % paraformaldehyde for 20 min, permeabilized with 0.5 % Triton X-100 in PBS for 5 min in 4 °C and washed with PBS. Fixed cells were incubated in primary antibody solution overnight at 4 °C, washed with PBS, and incubated for 1 h with secondary antibody solution at room temperature and washed again in PBS and water. Coverslips were mounted on glass slides using DABCO mounting medium (Fluka) with DAPI. Staining was visualized on a Zeiss 510 Meta confocal microscope using the 63X objective. Images were processed with ImageJ and Zeiss ZEN 2008 software for adjusting contrast, brightness, and size.
All antibodies were diluted in 1 % FBS in PBS. The primary antibodies used for staining were mouse anti-lamin A/C (Jol2, 1:20), rabbit anti-emerin (1:30, Proteintech), and rabbit anti lamin C (C1, 1:20, donated by Professor C.J. Hutchison). The secondary antibodies were donkey anti-mouse conjugated with TRITC (1:50), donkey anti-rabbit conjugated with Cy5 (1:50), and donkey anti-mouse conjugated with Daylight 649 (1:100), all purchased from Jackson ImmunoResearch.