TUNEL Assay for Apoptosis Detection

TUNEL assay was used to detect in situ apoptosis. The assay was performed using the In Situ Cell Death Detection (TUNEL) Kit with TMR Red (Roche, 12156792910) as previously described ^{12 (link)}. Briefly, 30 μm thick PL frozen sections were post-fixed with 1% PFA for 20 min at 22–24 °C and rinsed with PBS (three times, 5 min each). Sections were then permeabilized in 0.1% sodium citrate and 1% Triton X-100 for 1 h at 22–24 °C. After rinsing in PBS (three times, 5 min each), sections were incubated with TUNEL reaction solution according to the vendor’s instructions. Incubation was performed in a humidified chamber for 3 h at 37 °C in the dark. Sections were rinsed and mounted with Fluoromount containing DRAQ-5 (1:1000). As positive controls, sections were treated with DNase I (10 U/mL, New England Biolabs, M0303S) for 1 h at 37 °C, rinsed in PBS (three times, 5 min each), and incubated with the TUNEL mixture.