An exploration of immunofluorescence protocol detecting co-localization of p53 and mitochondria_West China Medical Journal

Authors：

LONG Dan , FENG Li , CHEN Xuelu ,  LI Shengfu ,  ZHOU Yanni

Key Laboratory of Transplant Engineering and Immunology of the Ministry of Health, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

LI Shengfu, Email: lishengfu@126.com; ZHOU Yanni, Email: 812053414@qq.com

Keywords：

p53; Mitochondria; Co-localization; Immunofluorescence

DOI：

10.7507/1002-0179.201908146

Video：

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Objective To establish a better immunofluorescence protocol to detect co-localization of p53 and mitochondria which may benefit studies aiming to detect mitochondrial expression of proteins.Methods HeLa 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.Results The 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.Conclusions Co-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.

Citation： LONG Dan, FENG Li, CHEN Xuelu, LI Shengfu, ZHOU Yanni. An exploration of immunofluorescence protocol detecting co-localization of p53 and mitochondria. West China Medical Journal, 2020, 35(8): 948-953. doi: 10.7507/1002-0179.201908146 Copy

1.	Bonda E, Rahav G, Kaya A, <italic>et al</italic>. p53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA. Oncotarget, 2016, 7(45): 73323-73336.
2.	Castrogiovanni C, Waterschoot B, De Backer O, <italic>et al</italic>. Serine 392 phosphorylation modulates p53 mitochondrial translocation and transcription-independent apoptosis. Cell Death Differ, 2018, 25(1): 190-203.
3.	Park JH, Zhuang J, Li J, <italic>et al</italic>. p53 as Guardian of the mitochondrial genome. FEBS Lett, 2016, 590(7): 924-934.
4.	Li HS, Zhou YN, Li L, <italic>et al</italic>. HIF-1αprotects against oxidative stress by directly targeting mitochondria. Redox Biol, 2019, 25: 101109.
5.	Khan Z, Michalopoulos GK, Stolz DB. Peroxisomal localization of hypoxia-inducible factors and hypoxia-inducible factor regulatory hydroxylases in primary rat hepatocytes exposed to hypoxia-reoxygenation. Am J Pathol, 2006, 169(4): 1251-1269.
6.	Moutsatsou P, Psarra AM, Tsiapara A, <italic>et al</italic>. Localization of the glucocorticoid receptor in rat brain mitochondria. Arch Biochem Biophys, 2001, 386(1): 69-78.
7.	Sepuri N, Tammineni P, Mohammed F, <italic>et al</italic>. Nuclear transcription factors in the mitochondria: a new paradigm in fine-tuning mitochondrial metabolism. Handb Exp Pharmacol, 2017, 240(15): 3-20.
8.	Morohashi Y, Bewley JD, Edward CY. Biogenesis of mitochondria in imbibed peanut cotyledons: II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA. Plant Physiol, 1981, 68(2): 318-323.
9.	Frisell WR, Patwardhan MV, Mackenzie CG. Quantitative studies on the soluble compartments of light and heavy mitochondria from rat liver. J Biol Chem, 1965, 240: 1829-1835.
10.	Marchenko ND, Zaika A, Moll UM. Death signal-induced localization of p53 protein to mitochondria. J Biol Chem, 2000, 275(21): 16202-16212.
11.	Sansome C, Zaika A, Marchenko ND, <italic>et al</italic>. Hypoxia death stimulus induces translocation of p53 protein to mitochondria. FEBS Lett, 2001, 488(3): 110-115.
12.	Humpton TJ, Vousden KH. Regulation of cellular metabolism and hypoxia by p53. Cold Spring Harb Perspect Med, 2016, 6(7): 1-20.
13.	Leszczynska KB, Foskolou IP, Abraham AG, <italic>et al</italic>. Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT. J Clin Invest, 2015, 125(6): 2385-2398.
14.	Mori H, Cardiff RD. Methods of immunohistochemistry and immunofluorescence: converting invisible to visible. Methods Mol Biol, 2016, 1458(9): 1-12.
15.	Odell ID, Cook D. Immunofluorescence techniques. J Invest Dermatol, 2013, 133(1): e4.
16.	Dashzeveg N, Yoshida K. Cell death decision by p53 via control of the mitochondrial membrane. Cancer Lett, 2015, 367(2): 108-112.
17.	Murphy ME, Leu JI, George DL. p53 moves to mitochondria: a turn on the path to apoptosis. Cell Cycle, 2004, 3(7): 836-839.
18.	Wadhwa R, Taira K, Kaul SC. An Hsp70 family chaperone, mortalin/mthsp70/PBP74/Grp75: what, when, and where?. Cell Stress Chaperones, 2002, 7(3): 309-316.

1. Bonda E, Rahav G, Kaya A, <italic>et al</italic>. p53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA. Oncotarget, 2016, 7(45): 73323-73336.
2. Castrogiovanni C, Waterschoot B, De Backer O, <italic>et al</italic>. Serine 392 phosphorylation modulates p53 mitochondrial translocation and transcription-independent apoptosis. Cell Death Differ, 2018, 25(1): 190-203.
3. Park JH, Zhuang J, Li J, <italic>et al</italic>. p53 as Guardian of the mitochondrial genome. FEBS Lett, 2016, 590(7): 924-934.
4. Li HS, Zhou YN, Li L, <italic>et al</italic>. HIF-1αprotects against oxidative stress by directly targeting mitochondria. Redox Biol, 2019, 25: 101109.
5. Khan Z, Michalopoulos GK, Stolz DB. Peroxisomal localization of hypoxia-inducible factors and hypoxia-inducible factor regulatory hydroxylases in primary rat hepatocytes exposed to hypoxia-reoxygenation. Am J Pathol, 2006, 169(4): 1251-1269.
6. Moutsatsou P, Psarra AM, Tsiapara A, <italic>et al</italic>. Localization of the glucocorticoid receptor in rat brain mitochondria. Arch Biochem Biophys, 2001, 386(1): 69-78.
7. Sepuri N, Tammineni P, Mohammed F, <italic>et al</italic>. Nuclear transcription factors in the mitochondria: a new paradigm in fine-tuning mitochondrial metabolism. Handb Exp Pharmacol, 2017, 240(15): 3-20.
8. Morohashi Y, Bewley JD, Edward CY. Biogenesis of mitochondria in imbibed peanut cotyledons: II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA. Plant Physiol, 1981, 68(2): 318-323.
9. Frisell WR, Patwardhan MV, Mackenzie CG. Quantitative studies on the soluble compartments of light and heavy mitochondria from rat liver. J Biol Chem, 1965, 240: 1829-1835.
10. Marchenko ND, Zaika A, Moll UM. Death signal-induced localization of p53 protein to mitochondria. J Biol Chem, 2000, 275(21): 16202-16212.
11. Sansome C, Zaika A, Marchenko ND, <italic>et al</italic>. Hypoxia death stimulus induces translocation of p53 protein to mitochondria. FEBS Lett, 2001, 488(3): 110-115.
12. Humpton TJ, Vousden KH. Regulation of cellular metabolism and hypoxia by p53. Cold Spring Harb Perspect Med, 2016, 6(7): 1-20.
13. Leszczynska KB, Foskolou IP, Abraham AG, <italic>et al</italic>. Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT. J Clin Invest, 2015, 125(6): 2385-2398.
14. Mori H, Cardiff RD. Methods of immunohistochemistry and immunofluorescence: converting invisible to visible. Methods Mol Biol, 2016, 1458(9): 1-12.
15. Odell ID, Cook D. Immunofluorescence techniques. J Invest Dermatol, 2013, 133(1): e4.
16. Dashzeveg N, Yoshida K. Cell death decision by p53 via control of the mitochondrial membrane. Cancer Lett, 2015, 367(2): 108-112.
17. Murphy ME, Leu JI, George DL. p53 moves to mitochondria: a turn on the path to apoptosis. Cell Cycle, 2004, 3(7): 836-839.
18. Wadhwa R, Taira K, Kaul SC. An Hsp70 family chaperone, mortalin/mthsp70/PBP74/Grp75: what, when, and where?. Cell Stress Chaperones, 2002, 7(3): 309-316.

West China Medical Journal

An exploration of immunofluorescence protocol detecting co-localization of p53 and mitochondria

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