Lsm 5 image browser software

Immunofluorescence images were obtained using a Zeiss LSM 510 inverted confocal microscope and prepared for publication with Zeiss LSM 5 Image Browser software and Adobe Photoshop CS4.

Bladders were dissected and embedded in OCT compound for snap freezing (Tissue-Tek). Frozen blocks were sectioned at 10mm intervals using a Microm cryostat. For immunostaining, frozen tissue sections were fixed in 4% paraformaldehyde for 30min at 4°C. After washing three times with PBS, tissue sections were blocked in 2% goat serum in PBS containing 0.25% Trion X-100 for 1hr, and incubated with primary antibodies diluted in blocking solution overnight at 4°C in a humidified chamber (Rabbit anti-CK5, 1:300 dilution, ab53121 from Abcam; Rat anti-CK18/8, 1:300 dilution, Troma-I from hybridoma bank, Rabbit anti-GFP, 1:1000 dilution, A-11122 from Invitrogen). Sections were washed three times with PBS containing 0.25% Triton X-100, incubated with DAPI and appropriate Alexa fluor 488, 594, or 633 conjugated secondary antibodies diluted 1:1000 in blocking solution for 2hr at RT, washed again three times, and mounted on slides with Prolong Gold mounting reagent (Invitrogen). All images were obtained using a Zeiss LSM 510 inverted confocal microscope and prepared for publication with Zeiss LSM 5 Image Browser software and Adobe Photoshop CS3.