Alexa fluor 488 or 568 secondary antibodies

Histological sections (5–8 microns) were used for the immunofluorescence reaction. Antigen retrieval was performed using citric acid buffer followed by blocking for unspecific binding using 3% (w/v) bovine serum albumin (BSA) in 0.5% (v/v) Triton X-100 phosphate-Buffered Saline (PBS). The tissue sections were incubated overnight with primary antibodies against Acetyl-Histone H4 (Lys 5) (PA5-40085, ThermoFisher); Acetyl-Histone H4 (Lys 8) (9HCLC, ThermoFisher); Acetyl-Histone H4 (Lys 12) (D2W6O, Cell Signaling Tech, Danvers, MA) Acetyl-Histone H4 (Lys 16) (E2B8W, Cell Signaling Tech.). Alexa Fluor 488 or 568 secondary antibodies (Invitrogen, Carlsbad, CA, USA) was used, followed by the Hoechst 33342 (blue stain-Invitrogen, Carlsbad, CA, USA) nuclear staining. Single tissue section was collected from each mouse to quantify each histone. Images were taken using a ×40 objective and a QImaging ExiAqua monochrome digital camera attached to a Nikon Eclipse 80i microscope (Nikon, Melville, NY, USA). Cell counting was performed on the images (1–3 fields) using the ImageJ^® software (National Institute of Mental Health, Bethesda, Maryland, USA). Only epidermis was included in the scoring. Labeling index or % of positive cells was defined as the number of positive cells (i.e., histone-Alexa Fluor 488 or 568 nuclear stain) divided by the total number of cells (blue nuclear stain) ×100.

Cells were grown on glass coverslips, washed with PBS and fixed with 4% formaldehyde for 10 min. After washing with PBS, cells were incubated with methanol at −20 °C for 10 min. Cells were then washed twice with PBS and incubated with primary antibodies for 1 h at RT. Following the washing step, coverslips were incubated with anti-rabbit or anti-mouse AlexaFluor-488 or -568 secondary antibodies (Invitrogen) with DAPI (Invitrogen) for 1 h at RT, washed again with PBS and mounted using Prolong Gold Antifade (Thermo Fisher Scientific). The primary antibodies used: LC3B (D11) XP™ (1:200, 3868, Cell Signalling), gamma H2A.X (phospho-S140) (1:1000, ab22551, Abcam), 53BP1 (H300) (1:500, sc-22760, Santa Cruz), TOMM20 (1:1000, ab56783, Abcam).

Wandering third-instar larvae were dissected in room temperature Phosphate Buffered Saline (PBS, Invitrogen) or HL3 Ringer’s medium without Ca²⁺ for < 20 min., or when quantifying glial projections, in HL3 Ringer’s containing 1.5 mM Ca²⁺ for <10 min. prior to fixation. Filets were then fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 30 min., washed in PBS with 0.2% Triton X-100 (PBST), and blocked in PBST with 5% normal goat serum, 0.01% bovine serum albumin, and 0.01% sodium azide for up to 2 h at room temperature. Filets were incubated overnight at 4°C in primary antibody diluted in block. Primary antibodies used were mouse polyclonal αPak3 (gift of S. Bahri, 1:250), rabbit αHRP (Jackson, 1:100), mouse αDLG (4F3 supernatant, Developmental Studies Hybridoma Bank [DSHB], 1:100), rat αElav (7E8A10, DSHB, 1:100), mouse αSlit (C555.6D, DSHB, 1:50), and mouse (mAb 3E6) or rabbit αGFP (Invitrogen, 1:300). Filets were then washed in PBST and incubated with the species-appropriate Alexa Fluor 488 or 568 secondary antibodies (Invitrogen) diluted 1:300 in block, for 2 h at room temperature or overnight at 4°C. After further washing, filets were mounted in Vectashield (Vector Laboratories) or ProLong Antifade (ThermoFisher).