Sp5 dmi microscope

Adult eye sectioning was performed as previously described⁸⁶ .
Third larval instar eye discs were dissected in ice-cold PBS and fixed in PBT (PBS + 0.1% Triton-X)-4% formaldehyde for 12 minutes at room temperature. For immunohistochemistry, following primary antibodies were used: rat anti-DE-cad (1:20, DSHB), mouse anti-Fmi (1:10, DSHB), rabbit anti-β-gal (1:200, ICL), rat anti-Elav (1:100, DSHB), chicken anti-GFP (1:1000, Aves Labs). Secondary antibodies were obtained from Jackson Laboratories. Eye disc images were acquired by using Leica SP5 DMI microscope.

IHC images were obtained using a Hamamatsu NanoZoomer S210 digital slide scanner. Fiji ImageJ software was used to measure the level of immunostaining for KIBRA in glomeruli. Color deconvolution was selected and specified to DAB vectors. Images were converted to black-and-white and a threshold level was selected for each antibody and used for each image quantified. Glomerular regions were selected for measurement of percentage area with positive staining based on the set threshold level for each antibody. At least 35 glomeruli were selected and quantified per mouse.
Immunofluorescence images were obtained using a Leica SP5 DMI microscope. Imaris software was used to measure the level of immunostaining for YAP. Surfaces were created for DAPI- and synaptopodin-labeled areas, and a threshold value for YAP staining was selected, whereby mean intensity measured above this threshold was considered “positive” and below was considered “negative” for YAP staining. The mean intensity of YAP staining within DAPI-designated surfaces colabeled for synaptopodin on the periphery of each glomerulus was also quantified, representing nuclear YAP expression in podocytes. At least 5–6 glomeruli were quantified per mouse and a total of 4 mice were quantified per group (ADR-treated controls and KIBRA-OE mice).