Immunostaining of Drosophila Tissues

Immunostaining was performed based on previous publication [15 (link)]. In brief, tissues were first dissected in 1X PBS (for guts) or in 4%formaldehyde (for thoraces), then fixed for 45 min at room temperature in 4%formaldehyde. After wash for 1 h in washing buffer (PBS, 0.5% BSA, 0.1% Triton X-100), tissues were incubated with primary antibodies and secondary antibodies diluted in washing buffer. Samples are then mounted and imaged with Zeiss AxioImager M2 with the apotome system. Images were then processed with ZEN and Image J software. Antibodies used in the studies:
rabbit anti-pH3 (Cat#06-570, Sigma-Aldrich) 1:1000,
rabbit anti-CREB (Cat#9197, Cell Signaling Technology), 1:800,
mouse anti-FK2(Cat#ENZ-ABS840-0100, Enzo Life Sciences),1:300,
mouse anti- Prospero (Cat# MR1A, Developmental Studies Hybridoma Bank, DHSB),1:100
rabbit anti-phospho-eIF2α(#Y407807, Applied Biological Materials),1:400,
anti-GAPDH (YEAEN, Cat#30210ES60) 1:2000.