Tunel kit with tmr red

The In Situ Cell Death Detection (TUNEL) Kit with TMR Red (Roche, 12156792910) was used to detect in situ apoptosis. Forty-micrometers cryosections were re-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 instruction, i.e., incubated in a mixture of 25 μL of terminal-deoxynucleotidyl transferase solution and 225 μL of label solution. 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). For a positive control, sections were treated with DNase I (10 U/mL, New England Biolabs, M0303S) for 1 h at 37 °C and rinsed in PBS (three times, 5 min each), followed by incubation with the TUNEL mixture.

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.