Tn-Mediated Histolysis in Drosophila Development

To examine the effect of Tn in DEOM histolysis, white prepupae at 0 h were collected and aged until 8 h APF, 12 h APF, or 24 APF. Muscle preparations were dissected, fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 30 min and immunostained as indicated^{75 (link)}. To analyze Tn function in salivary gland and midgut histolysis, wandering L3 larvae were dissected, fixed with 4% formaldehyde in PBS, and stained with DAPI (4′,6-diamidino-2-phenylindole) and/or phalloidin. The following primary antibodies were used: rabbit anti-Caspase-3 (1:100, Cell Signaling Technology, Danvers, MA,USA), mouse anti-DIAP1 (1:200, B. Hay)^{76 (link)}, guinea pig anti-p35 (1:10, P. Meier)^{40 (link)}, and guinea pig anti-Tn^{48 (link)}. Secondary antibodies used for fluorescent immunolabeling were Alexa Fluor anti-mouse 488, Alexa Fluor anti-rabbit 488, Alexa Fluor anti-mouse 594, and Alexa Fluor anti-guinea pig 488 (1:400, Molecular Probes, Eugene, OR, USA). Phalloidin 488 and 594 were used for F-actin labeling (1:400, Molecular Probes, Eugene, OR, USA). Immunostained preparations were imaged on a Zeiss LSM 700, processed using the Zeiss Zen software and assembled into figures in Photoshop Elements.