Mouse anti phosphohistoneh3

Testes were dissected in Ringers solution, then fixed in 4% formaldehyde in Buffer B (75 mM KCl, 25mM NaCl, 3.3mM MgCl₂, 16.7mM KPO₄) plus 0.1% Triton X-100 for 20 minutes. Testes were washed in PBTx (1X PBS, 0.1% triton X-100), blocked (4% normal donkey serum in PBTx), and stained with primary antibodies overnight at 4°C. Primary antibodies used were mouse anti-fasciclin 3 (1:20, DSHB), goat anti-vasa (1:200, Santa Cruz sc-26877), rabbit anti-vasa (1:200, Santa Cruz, sc-30210), guinea pig anti-Tj (1:10,000, gift from Dorothea Godt [40 (link)]), rabbit anti-zfh1 (1:5000, gift from Ruth Lehmann [41 (link)]), rat anti-Ecadherin (1:20, DSHB), mouse anti-phosphohistone H3 (1:500, AbCam 14955), chicken anti-GFP (1:1000, AbCam 13970), rabbit anti-GFP (Invitrogen A6455) rabbit anti-Phospho-p44/42 MAPK (dpErk) (1:500, Cell Signaling 9101), rabbit anti-phospho4E-BP1 (Cell Signaling 2855) and mouse anti-Eya (1:20, DSHB). Samples were washed in PBTx, then incubated in secondary antibodies for at least 1 hour. All secondary antibodies were from Jackson ImmunoResearch (1:400). Samples were stained with Hoechst 33342 (1 ug/mL), and washed in PBTx. Testes were mounted on slides in 90% glycerol, 1X PBS with 2% N-propyl gallate anti-bleaching agent and stored at −20°.

Tissues were dissected from 3rd instar larvae were fixed and stained as ref.53 (link) with minor modifications. After fixation for 20 min in 4% (w/v) paraformaldehyde in PBS, dissected brains from third instar larvae were washed in PBS with 0.1% Triton-X (PBST), then blocked for 2 h in PBST with 5% FCS (blocking solution). Primary antibody staining was done overnight at 4ºC in blocking solution, washed three times with PBST and incubated with secondary antibody in blocking solution for 2 h at room temperature. After three washes in PBST, brains were mounted in Vectasheild mounting media (Vectorlabs). Primary antibodies were as follows: mouse anti-phospho-Histone H3 (Abcam, 1:500); rabbit anti-Laminin (1:1000); guinea-pig anti-Repo (1:1000); rabbit anti-Repo (1:25,000); mouse anti-NimC1 P154 (link)(1:30), mouse anti-Mmp1 (1:1:1 mix of 3A6B4, 3B8D12, 5H7B11 from DSHB diluted 1:10); mouse anti-β–gal (Promega, 1:100); mouse anti-SRF (Active Motif, 1:100). Secondary antibodies were conjugated to Alexa-Fluor 555 or 633 (Invitrogen, 1:500). TO-PRO-3 Iodide (Invitrogen, 1:1000) or DAPI was used to visualise DNA.