Alexa fluor 488 568 633

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.

Cells seeded on coverslips were fixed, and immunofluorescence staining was performed as previously described^{69 (link)}. The primary antibodies included anti-α-tubulin (GTX112141, GeneTex, Hsinchu, Taiwan or T5168, Sigma), anti-γ-tubulin (T6557 or T3559, Sigma), anti-pericentrin (ab4448 or ab28144, Abcam, Cambridge, UK) and anti-TOMM20 (ab56783, Abcam). Alexa-Fluor 488-, 568-, 633-, and 647-conjugated goat anti-mouse, as well as anti-rabbit IgG were purchased from Invitrogen (Carlsbad, CA, USA). Confocal images of the immunostained samples were obtained with a Leica TCS-SP5 microscope equipped with a HCX PLAPO ×63/1.4 objective at 300 Hz scanning speed; image stacks of 15–20 μm were collected with a 0.5-μm step size. The confocal image stacks were processed and maxima-projected in ImageJ for presentation.