Fig. 4.

Localization of pMLKL in vitrified-warmed oocytes from young and aged mice. a Specificity of anti-pMLKL antibody was confirmed in L929 cells treated with necroptosis-inducing reagents. -, no treatment; TSZ, a mixture of TNFα, LCL161, Z-VAD-FMK. Scale bar represents 50 μm. b Immunofluorescence staining of pMLKL in oocytes from young and aged mice before (fresh) and after vitrification-warming (V/W). Oocytes were fixed, permeabilized, and treated with anti-pMLKL antibody at 1:100 (green). DNA was counterstained with TOPRO-3-iodide (1:250). Experiments were repeated three times, using more than 14 oocytes per group. White scale bar represents 20 μm. c A representative image showing co-localization of pMLKL and pericentrin. The white boxed area is enlarged four times. The primary antibodies used are, anti-pMLKL (1:100, green) and anti-pericentrin (1:500, red). The arrow indicates the spindle pole and the arrowhead indicates kinetochore. White scale bar represents 10 μm