Mouse anti nc82

The antibodies used for ChIP were: H3 (Abcam ab1791, lot no. GR177884-2), H3K27ac (Abcam ab4729, lot no. GR200563-1), H3K27me3 (Millipore 07–499, lot no. 2475696), H3K36me3 (Abcam ab9050, lot no. GR204353-1), H3K4me3 (Abcam ab8580, lot no. GR190202-1), and H3K9me3 (Abcam ab8898, lot no. GR186864-1), and were validated by the company for specificity. For the H3K4me3 antibody, abcam reports strong binding to H3K4me3 but some cross reactivity with H3K4me2 [103 ]. The following primary antibodies were used for immunofluorescence: mouse anti-Nc82 (1∶20; Developmental Studies Hybridoma Bank) and rat anti-Fru^M (1:200) [18 (link)]. The secondary antibodies used for immunofluorescence were Alexa Fluor goat anti-rat 488 (1:1000), goat anti-mouse 633 (1:500), rabbit anti-GFP 488 conjugate (1:600), and streptavidin 488 conjugate (1:4000) (Thermo Fisher).

Immunostaining was performed as previously described in [11 (link)]. Briefly, fly heads were fully dissected and fixed in 400 µL of 4% paraformaldehyde (PFA), 0.3% Triton X-100 for 20 min on ice. Once fixation was completed, three quick washes followed by three 20-min washes were performed in PBS, 0.3% Triton X-100. Blocking was achieved with 1 h incubation in 5% normal goat serum (NGS), PBS, 0.3% Triton X-100 at room temperature on a rocking platform. Brains were incubated with the primary antibodies for two nights at 4°C on a rocking platform. Brains were then washed and incubated with the secondary antibodies overnight at 4°C. Vectashield (Vector Laboratories) was used as slide mounting medium. Antibodies used in this study and dilutions were as follows: Mouse anti-nc82 (mouse, Developmental Studies Hybridoma Bank, 1:100), Rabbit anti-GFP (G10362, Invitrogen. 1:500), Goat anti-Rabbit IgG Alexa Fluor 488 1:200 (A11008, Thermo Fisher, 1:200) and Goat anti-mouse IgG-Alexa Fluor 633 1:200 (mouse, A21052, Thermo Fisher, 1:200).

Adult fly brains were dissected, fixed, and stained using standard protocols. Briefly, tissue was dissected in phosphate-buffered saline (PBS), fixed in 4% PFA in PBL (PBS and 0.12M lysine) for 30 min at room temperature, washed 3× for 5 min in PBT (PBS and 0.5% Triton X-100), and blocked for 15 minutes in 10% normal goat serum in PBT (Sigma Aldrich, cat# G9023; St. Louis, MO, USA). Samples were then incubated with primary antibodies for 72 h at 4°C. After incubation, they were washed 3× for 10 min in PBT and incubated with secondary antibodies for 72 h at 4°C. Finally, the samples were washed 3× for 10 min in PBT and mounted in Vectashield (Vector Laboratories, cat# H-1000; Burlingame, CA, USA). As primary antibodies, we used: rabbit anti-GFP (1:2000, Molecular Probes, cat# 11122; Eugene, OR, USA), chicken anti-GFP (1:2000, Molecular Probes, cat# A10262), rabbit anti-DsRed (1:2000, Molecular Probes, cat# 710530), and mouse anti-nc82 (1:10, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). The secondary antibodies used were anti-rabbit or anti-chicken IgG conjugated to Alexa 488, anti-mouse or anti-rabitt IgG conjugated to Alexa 594, and anti-mouse IgG conjugated to Alexa 405. All microscopy of immunostainings was performed with a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany). Images were processed with ImageJ.