Dmr microscope

HeLa and HEK 293 were transfected with 2.0 μg of the plasmids using a calcium phosphate protocol as above. Forty-eight hours posttransfection, cells were rinsed with PBS and fixed in 4% paraformaldehyde, 1X PBS for 30 minutes at room temperature. The cells were washed twice with 10mM glycine in PBS and permeabilized with 1% Triton X-100 in PBS for 5 minutes. After 2 washes, cells were blocked in 3% BSA in 1x PBS for 1 hour at room temperature. The coverslips were then inverted over the primary antibodies and incubated for 1 hour in a humidified chamber. The cells were washed again and incubated with fluorescently labeled secondary antibodies for 1 hour. Following washing, the coverslips were incubated with 0.05 μg/ml of DAPI in PBS for 5 minutes to allow detection of the nuclei. Samples were mounted onto slides using a solution of 2-phenylenediamine in 100mM Tris pH 8.0, 90% glycerol. Images were obtained using OpenLab software on a Leica DMR microscope and captured with a Hamamatsu CCD camera. Anti-myc antibody was obtained from Invitrogen. Anti-SRm300 was provided by B. Blencowe. Staining for the HA epitope used supernatant from the 12CA5 hybridoma.

A Leica inverted DM Rb microscope attached with a Moticam Camera interfaced with Motic Images 2.0 software was used for the imaging of C. albicans filaments on plates. A Leica DMR microscope fitted with a Hamamatsu digital camera interfaced with Metamorph software was used to take microscopic images of C. albicans cells. For the d-Tomato red fluorescent signal, the rhodamine filter was used. All images were processed using Adobe Photoshop.