Ds126201 eos 5d mark 2

Malpighian tubules from aged and treated flies as indicated were manually micro-dissected in 1× PBS, washed and equilibrated into a 3:1 1× PBS:glycerol solution as in [30 (link)] and photographed on a Leica MZ FLIII Fluorescence Stereomicroscope with Leica MZ series 10×/21B Widefield adjustable eyepieces equipped with a Canon DS126201 EOS 5D MARK II camera, using visible light. Canon raw files (CR2) were converted into TIF format using the Adobe Lightroom 3.2 software (Adobe Systems, San Jose, CA, USA).

Micro-dissected Malpighian tubules were photographed on a Leica MZ FLIII Fluorescence Stereomicroscope with Leica MZ series 10X/21B Widefield Adjustable eyepieces equipped with a Canon DS126201 EOS 5D MARK II, using visible light. Canon raw files were converted into TIF format (Adobe Lightroom 3.2). Images were merged and processed with Adobe Photoshop CS5. Immunofluorescence: dissected Malpighian tubules were fixed (20 min. in 4% paraformaldehyde 1X PBS 0.1% Tween-20), washed in PBT, incubated with primary antibody (anti-BicC [24 (link)] 1:1000, anti-d-Myc monoclonal 1:20, gift of Dr. Bruce Edgar) followed by Alexa Fluor 488 or 546 conjugated secondary antibodies (Molecular Probes) and DAPI, mounted (ProLong Gold, Molecular Probes), and imaged (Zeiss LSM710 confocal microscope). Acquired images were exported as TIF files (ZEN 2012), and processed with Adobe Photoshop CS5. 5μm thick section of paraffin-embedded, formaldehyde-fixed kidneys from adult mice were de-paraffinized, treated with NaBH₄ for 30 min, washed with TBS, 1% SDS (5 min) and washed as described above prior to blocking (mouse IgG) and probing with 1:30 anti-Bicc1 polyclonal (Aviva Systems Biology), or blocking buffer (control), followed by Alexa-Fluor 546-conjugated secondary antibody and DAPI. Sections were mounted in ProLong Gold. Antibody controls are shown in S5 Fig.