Rabbit anti ph3 s10 and s28

Immunofluorescence: Tissues were dissected in PBS and fixed for 30–40 min in 4% para-formaldehyde (Polysciences, Inc.). After fixation samples were washed 3 times in PBS + 0.1% Triton X-100 (PBST) and incubated in primary antibodies overnight at 4°C. Samples were then washed as described and subjected to secondary antibody staining for 2 hr at room temperature followed by washing and mounting on Vectashield containing DAPI (Vector Laboratories, Inc.). Primary and secondary antibodies were incubated in PBST+ 0.5% BSA. Rabbit anti-pH3 S10 and S28 1:100 (Cell Signaling); rabbit anti-Bursicon (α-subunit) 1:250 (Luan H, Lemon WC, Peabody NC, 2006). Anti-mouse or anti-rabbit secondary antibodies Alexa 488 or Alexa 594 (Invitrogen) were used at 1:200 or 1:100, respectively. F-Actin was visualized with Alexa 488-Phallodin (Invitrogen) 1:500. Nuclei were counterstained with DAPI. Confocal images were collected using a Zeiss 710 Confocal microscope and processed with Zeiss ZEN 2010, ImageJ or Adobe Photoshop CS.

The primary antibodies used were as follows: chicken anti-GFP, 1:4,000 (Abcam); mouse anti-Delta, 1:20 (Developmental Studies Hybridoma Bank; DSHB); mouse anti-Prospero, 1:30 (DSHB); rabbit anti-pH3 S10 and S28, 1:100 (Cell Signaling); rabbit anti-Bursicon (α subunit), 1:250; and rabbit anti-β-gal 1:1,000 (Cappel).