ObjectiveTo establish a better immunofluorescence protocol to detect co-localization of p53 and mitochondria which may benefit studies aiming to detect mitochondrial expression of proteins.MethodsHeLa cells were treated with hypoxia and the expression of p53 was detected by immunoblotting. HeLa cells were fixed with methanol, methanol: acetone (1: 1, v/v) mixture, and 4% paraformaldehyde, respectively; the former two groups were not permeable, while the latter was penetrated with 0.1% Triton-X 100 and stained with p53 and mitochondria at the same time. After HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 was reduced to 0.05%, 0.025%, 0.01%, and 0.005%. After the HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 decreased to 0.01% and 0.005% for the first time, then, after staining with p53, the mitochondria were stained with 0.1% Triton-X 100 for the second time.ResultsThe expression of p53 was up-regulated (P<0.01) after hypoxia, which could be used in the following immunofluorescence experiment. The co-localization of p53 and mitochondria was observed in the nucleus and cytoplasm in both the methanol group and the mixed solution group. The co-localization of p53 was the most obvious in the mixed solution group. After using 0.1% Triton-X 100, the p53 signal was mainly in the nucleus, but no co-localization was observed. After fixation with 4% paraformaldehyde, to some extent, the reduced concentration of 0.05% and 0.025% Triton-X 100 weakened the p53 signal in nucleus and enhanced the co-localization signal. However, the signal in nucleus was still stronger than that in cytoplasm. When it was reduced to 0.01% and 0.005%, p53 signal was detected in cytoplasm but not in nucleus, suggesting that the nuclear membrane was not penetrated under this condition, but it also failed to penetrate the mitochondrial membrane, leading to the failure of mitochondrial labeling. The second permeability completely avoided the p53 signal in nucleus, and successfully labeled mitochondria, and the co-localization of p53 and mitochondria was detected.ConclusionsCo-localization of p53 and mitochondria is detectable in cells fixed by methanol or methanol and acetone mixture which brings out better results. Penetrating twice with Triton X-100 of different concentrations following paraformaldehyde fixation help avoid signals in nuclei and falicitate co-localization detection.