Mouse anti disc large

Imaginal discs of third instar larvae were dissected in PBS and fixed for 20 min in 4% PFA/PBS. Subsequently, discs were washed three times in PBS + 0.2% Triton X-100 and blocked with 1% BSA for 1 h, incubated over night with primary antibodies in PBS + 0.2% Triton X-100 + 1% BSA, washed three times and incubated for 2 h with secondary antibodies. After three washing steps and DAPI-staining, discs were mounted with Mowiol. Embryos were fixed and stained as described before [32 (link)]. Primary antibodies used were as follows: anti Baz (1:500) [32 (link)], goat anti GFP (1:500, #600-101-215, Rockland, Pottstown, PA, USA), mouse anti Disc Large (1:100, 4F3, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA) and mouse anti Histone-3 phospho-S10 (1:500, Cell Signaling #9706, Danvers, MA, USA). Secondary antibodies conjugated with Alexa 488, Alexa 568 and Alexa 647 (Life Technologies, Carlsbad, CA, USA) were used at 1:400. Images were taken on a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) using lightning program and processed using ImageJ version 1.53t.

Brain tissue was dissected in ice-cold 1X PBS and fixed with PLP (8% paraformaldehyde in NaOH and PBS with lysine (1)-HCl) for one hour at room temperature as described in [65 (link)]. After fixation, the tissue was washed twice for 15 minutes with PBS-0.5% Triton X and then incubated for one hour in blocking solution (20% donkey serum, 0.5% Triton X in PBS) at room temperature. The primary antibody, mouse anti-disclarge (Developmental Studies Hybridoma Bank, University of Iowa, USA) was used at a 1:200 dilution and incubated overnight at 4° Celsius in blocking solution. After washing twice with PBS-0.5% Triton X, the tissue was incubated with the secondary antibody, 1:200 anti-rat-CY3 (Dianova, Hamburg, Germany). The brains were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and scanned using confocal microscopy with a Leica SP5-2. The images were analyzed using the StackGroom plugin in ImageJ [66 ].