Anti rat alexa fluor 555

Cells were pulse-labelled with 50 μM ldU for 20 minutes. They were then incubated with 4 mM HU for 4 hours and followed by 40 minutes in medium containing 100 μM CIdU. The cells were harvested, lysed in 200 mM Tris-HCl pH 7.4, 0.5% SDS, 50 mM EDTA, and the DNA fibers were spread on glass slides. The slides were incubated with 0.5mg/ml pepsin in 30 mM HCl at 37°C for 20 minutes, the DNA was denatured in 2.5 M HCl for 1 hour and blocked with 1% BSA containing 0.1% Tween 20 in PBS. The nucleotide analogues were detected with primary antibodies against CldU (Novus), and IdU (BD Biosciences) and the secondary antibodies anti-rat Alexa Fluor 555 and anti-mouse Alexa Fluor 488 (Thermo Fischer Scientific). Coverslips were mounted on slides in Dako Fluorescence Mounting Medium (Dako). Images were captured with a Nikon Ni-E microscope and a DS-Qi2 camera equipped with a 20X objective and analyzed with NIS-Elements AR imaging software.

Brains (c564>fs(1)h-IR, n=8; c564>0, n=5) were dissected from adult male flies starved for 1 h into 1× PBS. Dissected brains were fixed in 4% formaldehyde (methanol-free) 0.1% Triton X-100 PBS (PBST) for 30 min at room temperature. Samples were then rinsed twice with 0.1% PBST and washed four times for 1 h. 5% normal goat serum in 0.1% PBST was used as a blocking agent for 1 h at room temperature. Brains were then incubated overnight at 4°C with primary antibodies in 5% normal goat serum in 0.1% PBST. The primary antibodies used were: anti-rabbit-dILP2 (gift from Nazif Alic, University College London, London, UK; 1:1000) and anti-Rat-Elav (DSHB #7E8A10; 1:1000). Brains were rinsed twice with PBST, and washed four times with 0.1% PBST for 1 h. Secondary antibodies were diluted in 5% normal goat serum in 0.1% PBST and incubated with the brains for 1 h at room temperature. The secondary antibodies used were: anti-rabbit-Alexa-Fluor-488 (Thermo Scientific #A27034; 1:500) and anti-rat-Alexa-Fluor-555 (Thermo Scientific #A21434; 1:500). Samples were then rinsed twice with 0.1% PBST, and washed four times for 1 h. Brains were mounted on glass coverslips in Vectashield (Vector Laboratories). All incubations and washes were performed in a rotator. Images were acquired using a Zeiss LSM 510 confocal microscope and edited using Fiji/ImageJ.

Embryos were stained with an antibody against endomucin according to Vieira et
al.70 (link). In brief, embryos were fixed for two hours on ice
with PBS containing 4% PFA. After three washing steps with PBS containing 0.1%
Triton X-100 and blocking of unspecific binding sites by incubation with PBS
containing 10% FCS and 0.1% Triton X-100 for 30 minutes at room temperature,
embryos were incubated with rat monoclonal antibody against endomucin (1:100,
Santa Cruz Biotechnology) dissolved in PBS over night at
4 °C followed by five washing steps with PBS at room
temperature. After incubation with secondary antibody anti-rat Alexa Fluor-555
(1:200, Molecular Probes) over night at 4 °C and three
further washing steps with PBS, embryos were mounted and analyzed by laser
scanning confocal microscopy (SP5; Leica, Wetzlar, Germany). Staining of whole
embryos for LacZ was as described previously71 .