Apoptag kit

The TUNEL assay was performed using the ApopTag Red in situ apoptosis detection kit (Millipore) according to the manufacturer's instructions. Fifteen min after heat shock induction, wing discs were dissected in 1xPBS and fixed in 1xPBS with 4% paraformaldehyde at the room temperature. After fixation, samples were washed with PBS 0.1% Triton-X100 and incubated in equilibration buffer (Apop Tag kit; Millipore) for 10 s. Then, samples were incubated in reaction buffer (TdT enzyme; ratio 7:3; Apop Tag kit) at 37°C for 1 h. The TdT reaction mix was replaced with stop buffer (diluted 1:34 in dH₂O; Apop Tag kit) and incubated for 10 min at RT. Samples were then washed with PBS 0.1% Triton-X100 three times and incubated with anti-digoxigenin antibody solution (diluted 31:34 in blocking solution; ApopTag kit) and anti-cDCP1 antibody overnight at 4°C. Samples were then washed with PBS 0.1% Triton-X100 three times again and incubated with the secondary antibody for 3 h at room temperature, followed by washing with PBS 0.1% Triton-X100 thee times and mounted on a glass slide.

The TUNEL assay was performed using the ApopTag Red in situ apoptosis detection kit (Millipore) as described previously (Ciesielski et al, 2022 (link)). Wandering 3^rd instar larvae were dissected in 1×PBS and fixed for 30 min in 1×PBS with 4% paraformaldehyde at room temperature (RT). After fixation, samples were washed with PBS 0.1% Triton‐X100 and incubated in equilibration buffer (Apop Tag kit; Millipore) for 10 s. Then, samples were incubated in reaction buffer (TdT enzyme; ratio 7:3; Apop Tag kit) at 37°C for 1 h. The TdT reaction mix was replaced with stop buffer (diluted 1:34 in dH2O; Apop Tag kit) and incubated for 10 min at RT. Samples were then washed with PBS 0.1% Triton‐X100 3 times and incubated with antidigoxigenin antibody solution (diluted 31:34 in blocking solution; ApopTag kit) overnight at 4°C. Samples were then washed with PBS 0.1% Triton‐X100 3 times again and mounted on the slide glass.

WDs were fixed in 4% PFA for 20 min, washed twice in PBS (2 × 5 min), washed twice (2 × 10 min) in 1xBSS (5xBSS: 270 mM NaCl, 200 mM KCl, 37 mM MgSO₄, 12 mM CaCl₂.2H₂O, 24 mM tricine, 1.8% glucose, and 8.5% sucrose), followed by two washes (2 × 5 min) in PBTw (0.1% Tween 20 in PBS). The samples were then refixed in 4% PFA for 20 min, washed five times (5 × 5 min) in PBTw, incubated in equilibration buffer (ApopTag kit; Millipore) for 1 h, followed by ON incubation with the TdT enzyme in reaction buffer (ratio 3:7; ApopTag kit) at 37 °C. The reaction was stopped by replacing the reaction mix with stop buffer (diluted 1:34 in dH2O; ApopTag kit) and incubation for 3-4 h at 37 °C. The samples were then washed three times (3 × 5 min) in PBTw, blocked in BTN solution (1xBSS, 0.3% Triton X-100, and 5% normal goat serum) for 1 h at RT, and incubated ON in the dark with anti-digoxigenin antibody solution (diluted 47:53 in blocking solution; ApopTag kit) at 4 °C. Samples were then washed four times (4 × 20 min) in 1xBSS, and mounted in Fluoromount-G (SouthernBiotech).
TUNEL labeling of embryos was performed following a modified manufacturer’s protocol^{69 (link)}. To induce apoptosis in germ cells we used embryos laid by nos-Gal4 females mated to UAS-hid males.