Apoptag in situ apoptosis detection kit

TUNEL staining (MilliporeSigma ApopTag In Situ Apoptosis Detection Kit) of mouse thyroid was performed in tissue sections, and nuclei were counterstained with DAPI. Quantitation of images was performed both manually and by using ImageJ (NIH) with the ITCN plugin.

TUNEL assay was performed using ApopTag in situ apoptosis detection kit (Millipore-Sigma). Briefly, Acta2-CreER^T2, ROSA26R^(Zs/+) mice were injected with 4-OH TM (200 µg per day) at P6.5, P8.5 and P10.5. Lungs were harvested on P11.5, P13.5, P14.5, P16.5 and P18.5 and P30.5 and fixed in 4% PFA. Cyrosections (10 µm) were treated with pre-cooled ethanol:acetic acid (2:1) for 5 min at −20°C and washed in PBS. Slides were then processed according to instructions of the manufacturer, culminating in incubation with anti-digoxigenin-rhodamine fluorochrome conjugate for 30 min at RT, protected from light. Sections were washed in PBS, mounted and directly imaged for Zs and rhodamine.

Because cleaved caspase‐3 expression was more robust than expected, a second method was used to identify apoptotic cell death and determine whether caspase‐3 expression was associated with apoptosis. An ApopTag® In Situ Apoptosis Detection Kit (Millipore Sigma) was used to label DNA fragmentation, present in apoptotic cells. The kit stained both single and double stranded breaks that are associated with apoptotic cell death using the TUNEL assay technique. The assay was completed on one slide per animal per the directions provided in the kit. Sections were reacted in a DAB horseradish peroxidase (HRP) substrate, counterstained using Safranin‐O (Millipore Sigma), mounted with Permount (Thermo Fisher Scientific) medium, and coverslipped. Imaging and unbiased stereology was identical to methods described previously for caspase‐3, and area fractions for TUNEL positive cells were calculated for each animal. One section per animal was treated with DNase1, an enzyme that nonspecifically cleaves DNA, for 10 min at room temperature and served as a positive control. One section per animal was not treated with the terminal deoxynucleotidyl transferase (TdT) enzyme and served as a negative control.