Cf488a donkey anti rabbit igg

Platonin was synthesized by and obtained from Gwo Chyang Pharmaceuticals (Tainan, Taiwan; Figure 1A). Primary antibody against cleaved-caspase-3 was purchased from Abcam (Cambridge, UK). The anti-Iba1 antibody was purchased from Wako Chemicals USA (Richmond, VA, USA). The anti-NeuN antibody was from Merck Millipore (Darmstadt, Germany). For immunostaining, CF488A donkey anti-mouse IgG and CF488A donkey anti-rabbit IgG were purchased from Biotium (Fremont, CA, USA). Dihydroethidium (DHE) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Platonin was dissolved in phosphate-buffered saline (PBS) and stored at 4 °C.

Morphological and histological analyses of embryos were collected at various times of gestation. For histological analysis, embryos were rinsed with ice‐cold PBS and fixed with 4% paraformaldehyde at room temperature for 10–30 min, dehydrated through graded alcohols, and embedded in paraffin. Embryos were sectioned at 5 μm thickness and stained with haematoxylin and eosin. Sectioned embryos were photographed using a Nikon E800M inverted microscope. For immunofluorescent analysis, paraffin sections were rehydrated, immunostained with cleaved Caspase‐3 antibody (9664, Cell Signalling Technology), Ki‐67 antibody (12202, Cell Signalling Technology). Antigen retrieval was performed according to the individual antibody instructions. CF488A Donkey anti‐Rabbit IgG (20015, Biotium) and CF488A Donkey anti‐Mouse IgG (20,014, Biotium) were used as the secondary antibody. Nuclei were counterstained with 1 μg/ml Propidium Iodide (P4170, Sigma‐Aldrich), washed and mounted.
Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end‐labeling (TUNEL) assay was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (8445, MBL). Briefly, paraffin‐embedded sections were deparaffinized, DNA nick end‐labelled, and counterstained with 1 μg/ml Propidium Iodide and mounted. The slides were analysed using a TCS SP8 confocal microscope (Leica).