Ab821

Whole-mount in situ hybridization (WISH) was performed as previously described [31 (link)]. The primers used to amplify for probe synthesis are listed in Table A in S1 Table. Then PCR products were used to generate probes using the DIG RNA labeling Kit (Roche Applied Science, Penzberg, Germany). The WISH images were captured using the SteREO Discovery V20 microscope (Carl Zeiss, Germany).
Whole-mount antibody staining was performed as previously described [51 (link)]. Primary antibodies anti-pRS6 (S240/244) (1:500; #2215 Cell Signaling, USA), anti-LC3B (1:1000; ab192890, Abcam), anti-Dendra2 (1:1000, AB821, Evrogen, Moscow, Russia). Secondary antibodies used in the study are donkey anti-goat IgG Alexa fluor 568-conjugated (1:1000, Invitrogen) and donkey anti-rabbit IgG Alexa fluor 488-conjugated (1:1000, Invitrogen). Images were captured using ZEN2010 software equipped on an LSM880 confocal microscope (Carl Zeiss).

Antibody staining and TUNEL assay were performed as described previously (29 (link)). The following antibodies were used: antibodies against Dendra2 (1:1000; AB821, Evrogen, Moscow, Russia), phospho-4E-BP1 (Thr-37/46) (1:500; catalog no. 2855, Cell Signaling), Hnf4a (1:200; sc-6556, Santa Cruz Biotechnology, Inc.), Prox1 (1:500; ab5475, Chemicon), 2F11 (1:1000; ab71826, Abcam, Cambridge, MA), and PCNA (1:1000; SAB2701819, Sigma).

For whole-mount antibody staining, larvae were removed the skin with the Tweezers, washed several times with PT (PBS + 1% Triton X-100) and incubated with primary antibodies below: Anxa4 (1:1000; ab71286, Abcam, Cambridge, MA), 5-methyl-cytosine (1:100; ab10805, Abcam, Cambridge, MA), Alcam (1:50; zn5, ZIRC, Eugene, OR), Bhmt (1:500, a kind gift from Jinrong Peng, Zhejiang University, China), Dendra2 (1:1000; AB821, Evrogen, Moscow, Russia), pS6 (1:500; 2215, Cell Signaling, MA, USA), p4EBP1 (1:500; 2855, Cell Signaling, MA, USA), Dnmt1 (1:200, sc-20701, Santa Cruz Biotechnology, Santa Cruz, CA, a kind gift from Jingwei Xiong, Peking University, China), GFP (1:1000, ab6658, Abcam, Cambridge, MA), DsRed (1:500, sc-101526, Santa Cruz Biotechnology, Santa Cruz, CA) and Tomato (1:1000; orb182397, Biorbyt, TX, USA), After primary antibody incubation, larvae were washed several times with PT and incubated with secondary antibodies conjugated to Alexa Fluor 488/568/633 (1:1000; Invitrogen, Grand Island, NY). The primary and secondary antibodies were diluted in the blocking solution (PBS + 4% BSA + 1% Triton X-100) and incubated at 4 °C overnight and washed with PT for five times.