Tcs sp5 mp smd flim confocal laser scanning microscope

Brains were dissected from 3rd instar wandering larvae, fixed in 4% formaldehyde-PBS for 30 min at RT and washed in PBT buffer (0.3% Triton X 100 in PBS). Following blocking for 2 h at RT in 5% bovine serum albumin (BSA)/PBT, samples were incubated with respective primary antibodies at 4°C o/n. Samples were washed three times (15 min each) with PBT and incubated with respective secondary antibodies for 2 h at RT. After three washes in PBT, brains were mounted in Vectashield Mounting Medium (Vector laboratories). Fluorescence images were acquired using a Leica TCS SP5 MP SMD FLIM confocal laser scanning microscope. Antibodies used: rat anti-dILP2 [5] (link), rabbit anti-dILP2 [48] (link), guinea pig anti-dILP2 (see below), rabbit anti-dILP5 (5), anti-rat Alexa fluor 633 (Invitrogen), anti-rabbit Alexa fluor 633 (Invitrogen) and anti-guinea pig Alexa fluor 633 (Invitrogen). Confocal images of each IPC cluster were taken using the same scan and laser power settings. Total signal from each cluster was quantified using ImageJ software (NIH). Representative images were cropped and processed identically for all the samples of every experiment.

Brains were dissected from non-wandering third-instar larvae raised on standard laboratory diet, fixed in 4% formaldehyde in PBS for 30 min at room temperature, and washed in PBT buffer (0.3 % Triton X 100 in PBS). They were blocked using 5% BSA in PBT buffer for 2 h at room temperature. Samples were incubated with rabbit anti-dILP2 antibodies^{79 (link)} at 4 °C overnight. Samples were washed thrice in PBT (15 min each), incubated with goat anti-rabbit antibodies for 2 h at room temperature. Both antibodies were used in the dilution of 1:400. After three washes, they were mounted in Vectashield mounting medium (Vector Laboratories). Images were acquired using the same scan and laser power settings for each IPC cluster with a Leica TCS SP5 MP SMD FLIM confocal laser scanning microscope. Total signal from each cluster was quantified using ImageJ software (NIH).