Midguts were dissected from flies at 10, 20 and 40 days, fixed in 4% paraformaldehyde and stained as described [17 (link)]. Following washing, samples were mounted and imaged using the Leica camera attachment using LAS V4.1 software, or the Zeiss 780 combined confocal/FCS/NLO system, mounted on an inverted Axio Observer Z1. Fixed tissue was incubated overnight with mouse anti-gal 1:500 (Invitrogen); rabbit anti-pH3 1:300 (Invitrogen) or rabbit anti-INDY 1:300 [10 (link)] primary antibodies diluted in PBT [0.1%Triton X-100 in phosphate-buffered saline (PBS)] at 4°C. Following washing and blocking, tissue was incubated with the goat anti-rabbit Cy3 1:300 (Jackson) or goat anti-mouse FITC 1:300 (Jackson) secondary antibodies and DAPI 1:1000 (Invitrogen) diluted in PBT and 2% donkey serum for 1 hour at room temperature. Images were analyzed using Adobe Photoshop or Image J. Variability between different regions of the gut was reduced by quantifying images from the same designated region for each genotype in a 0.06x 0.02cm area. Cells were counted, values averaged and standard deviation calculated separately.
Free full text: Click here