Lsm 510 meta nlo confocal microscope

Cells were plated on coverslips (50,000 per coverslip) in a 24-well plate, allowed to attach for at least 4 hours, and transferred to control or transwell wells. After 48 hours, cell were stained with BODIPY (493/503) by washing with PBS, fixing with 3.7% formaldehyde at RT for 40 minutes, and incubating with BODIPY (493/503) (1:1000) at RT for 1 hour. Coverslips were washed and mounted onto slides using Vectashield with DAPI (Vector Laboratories). Images were taken using a Zeiss LSM 510 META NLO confocal microscope (Carl Zeiss AG, Göttingen, Germany) and a 40 × oil immersion lens. For CA9 staining, cells were washed with PBS and fixed with cold methanol. Coverslips were stained with rabbit monoclonal anti-CA9 antibody (1:50) at 4°C overnight. Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000) was used as a secondary antibody, and DAPI was used as a nuclear stain. Coverslips were mounted using Vectashield and imaged using a Zeiss LSM 510 META NLO confocal microscope using a 63 × oil immersion lens.

For immunofloresence analysis, cells were grown on coverslips and were transfected with the required plasmids. At the indicated time, cells were fixed with 3.7% formaldehyde and then stained with appropriate antibodies. Indirect immunostaining of cells and microscopy was carried out essentially as described previously [24 (link), 54 (link)]. All images were captured with LSM 510 Meta NLO Confocal Microscope from Carl Zeiss (Jena, Germany) using 63X oil immersion objective lens (NA 1.4). For colocalization, two, 0.33 μm, optical Z-sections through the centre of the cells were projected and was analyzed by using LSM 510 (version 3.2) software. Pearson’s correlation coefficients for colocalization were calculated by LSM 510 software. Nikon Eclipse Ni microscope (Japan) was also used for observing immunofluorescence. Images were captured using the Axiocam (Zeiss) CCD camera and processed with Axiovision 4 software. All images were assembled using Adobe Photoshop.