Apoptosis Detection in Tissue Sections

Immunohistochemical staining was performed using TACS 2 Tdt-Fluor In Situ Apoptosis Detection Kit (Trevigen; Burlington, Canada). Briefly, after sections were de-paraffinized, they were incubated with Proteinase K (1:50 in ddH₂O) for 30 min at 37 °C. Endogenous peroxidase quenching was performed for 5 min. Slides were coated in Labeling Reaction Mix at 37 °C for 1 h and immersed in 1× TdT Stop Buffer to stop the labeling reaction. Slides were then incubated with horseradish peroxidase-streptavidin (Vector Laboratories; Burlington, Canada), followed by Nova Red (Vector Laboratories) and haematoxylin (Sigma Aldrich). Immunofluorescent TUNEL staining was performed as previously [7 (link)], using a TMR-in situ cell death detection kit (Roche).