Method Article

Highlighting the Transition of Mitochondrial Cytochrome C by Immuno-transmission Electron Microscopy

DOI:

10.3791/68633

⸱

August 15th, 2025

 ,  ,  ,  ,  , 
1Medical Department, Hospital of Chengdu University of Traditional Chinese Medicine, 2Department of Critical Care Medicine, Traditional Chinese Medicine Hospital of Meishan, 3Sichuan Scientist Biotechnology Co., Ltd, 4Department of Critical Care Medicine, Deyang Hospital Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, 5Hospital of Chengdu University of Traditional Chinese Medicine, 6Department of Critical Care Medicine, Hospital of Chengdu University of Traditional Chinese Medicine

In This Article

Summary

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The protocol describes methods to study micromorphological changes in neurons and the transition of mitochondrial cytochrome c by immuno-transmission electron microscopy.

Abstract

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Protein location changes in the microstructure often present a precise regulation mechanism at the level of pathological organelles. How to accurately capture the location of this organelle protein transfer has positive significance and value for the diagnosis and treatment of diseases. Mitochondria play a central role in necrosis and the intrinsic pathway of apoptosis. Under normal conditions, cytochrome c (Cyt c) only exists between the inner and outer membranes of mitochondria and in the nucleus. When cells undergo apoptosis, the mitochondrial inner membrane permeability is altered, and Cyt c is released from mitochondria into the cytoplasm. In this study, a rat model of cerebral ischemia-reperfusion injury was established, and the micromorphological changes of neurons undergoing apoptosis were shown by immuno-transmission electron microscopy. Additionally, it shows the vision of immunogold labeling Cyt c transfer from the mitochondrial matrix to the cytoplasm after cerebral ischemia-reperfusion injury more precisely. The immuno-transmission electron microscopy technique presented in this protocol is suitable for the demonstration of any protein position transitions at the microscopic level.

Introduction

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According to the 2021 global burden of disease study, stroke was the 3rd leading cause of death and the 4th leading cause of disability-adjusted life-years. There were 11.9 million new stroke events, in which ischemic stroke constituted 65.3% (7.8 million)1. Neuronal death occurs after cerebral ischemia, including necrosis and apoptosis. The most severely affected brain tissue subsequently undergoes necrotic cell death. Neurons in the ischemic penumbra or periinfarct zone may undergo apoptosis after several hours or days2. In addition to bioenergetic and metabolic functions, mitochondria also play a....

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Protocol

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The animal program has been approved by the Ethical Committee of SLAS (IACUC Issue No: SYST-2024-018). Here, 6 pathogen-free male SD rats (200-220 g) were raised in independent ventilation cages, maintaining the laboratory environment at 22-25 °C and 45%-65% relative humidity. All rats underwent adaptive feeding for 1 week with free water and diet during the period.

1. Establishment of middle cerebral artery occlusion (MCAO) model

  1. Randomly divide the rats into the MCAO group and the sham operation group, with three rats in each group. See Figure 1 for the experimental flowchart....

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Results

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The neuroscore evaluation confirms the success of MCAO, according to Longa Score Scale, rats in MCAO group presented at least circling to the left (neuroscore 2). Rats of the sham operation group showed no obvious neurological dysfunction (neuroscore 0). No mortality was observed during the surgery day.The animals' neuroscore are listed in Table 1.

IEM analysis of the MCAO group revealed neurons with shrunken nuclei and elevated cytoplasmic electron density, accompanied by par.......

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Discussion

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The primary objective of reperfusion therapy in acute ischemic stroke is to restore blood perfusion to the ischemic region. Now we can achieve this goal through intravenous thrombolysis, endovascular treatment, and antithrombotic therapy. However, restoration of blood perfusion leads to ischemia-reperfusion injury, aggravates neuronal necrosis and apoptosis, leading to neurological impairment. During ischemia-reperfusion, abnormal leakage of electrons from the mitochondrial electron transport chain leads to the productio.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by the Special Scientific Research Project on Traditional Chinese Medicine of Sichuan Provincial Administration of Traditional Chinese Medicine(2021MS414).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.9% normal salineSichuan Kelun Pharmaceutical Co., Ltd.H20083400
200 Mesh formvar coated copper gridsZhongjingkeyi Technology Co., Ltd.BZ10262a
220-230g MCAO MonofilamentsBeijing Cinontech Co., Ltd.A4-243250
Absolute ethanolChengdu Kelong Chemical Co.,Ltd.2023101201
acetoneChengdu Kelong Chemical Co.,Ltd.2023120501
Animal anesthesia systemRayward Life Technology Co., Ltd.R500
Animal temperature maintainerRayward Life Technology Co., Ltd.69020
Baking tableShenzhen Bangqi Chuangyuan Technology Co., Ltd.CT-946
Bone shearFine Science Tools16102-11
capsular embedding moldsZhongjingkeyi Technology Co., Ltd.GP130-B
CentrifugeSCILOGEXD3024R
Conventional bright-field optical microscopyZeiss (Germany)Primo Star
Curved ophthalmic forceps (10 cm)Shanghai Medical Equipment Co., Ltd.211001
Diamond knifeDiatome (Switzerland)DU4530
Dissecting forcepsRayward Life Technology Co., Ltd.F12018-10
Dissecting forcepsRayward Life Technology Co., Ltd.F12010-10
Electric blast drying ovenShanghai YiHENG Scientific Instrument Co., Ltd.DHG-9030A
Electric shaverYiwu Kemei Electric Appliances Co., Ltd.KM-PG5002
Fixation fluidSichuan Scientist Biotechnology CO., Ltd.SC0001
glass slideNantong Mevid Life Science Co., Ltd.220518001
Gold-conjugated Goat anti-rabbit lgGSangon Biotech (Shanghai) Co.,Ltd.D111094
Hemostatic forceps (12.5 cm)Anqisheng Biotechnological Co., Ltd.PL068
Hemostatic forceps (14 cm)Anqisheng Biotechnological Co., Ltd.PL002
IsofluraneRayward Life Technology Co., Ltd.2024071201
L. R. White ResinTianjin Tainuo Science and Technology Co., Ltd.AGR1281A
Lead citrateSPI (US)GA10701
Needle holder Jiachi Medical Technology Co., Ltd.10044036898028
Osmium tetroxideTED PELLA (US)4008-220501
PBSServicebioG4202
Rabbit polyclonal antibody to cytochrome c  Affinity Biosciences, Ltd.AF0146
RatsBeijing Huafukang Biotechnology Co., Ltd.110322241102187881
SPI-Pon 812 embedding resin kitSPI(US)GS02660
StereomicroscopeSunny Optical Technology Co., Ltd.Soptop SZMN
Straight headed scissors (10 cm)Anqisheng Biotechnological Co., Ltd.PL063
Straight ophthalmic forceps (10cm)Shanghai Medical Equipment Co., Ltd.211001
Surgical scissorsRayward Life Technology Co., Ltd.S14001-10
Transmission electron microscopyHitachi (Japan), Ltd.HT7800
ultramicrotomeLeica(Germany)UC7
Uranium acetateSPI (US)GS02624

References

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  1. GBD 2021 Nervous System Disorders Collaborators. Global, regional, and national burden of disorders affecting the nervous system, 1990-2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet Neurol. 23 (4), 344-381 (2024).
  2. Taylor, R. C., Cullen, S. P., Martin, S. J.

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Tags

Mitochondrial Cytochrome CImmuno Transmission Electron MicroscopyCytochrome C ReleaseProtein LocationCerebral IschemiaApoptosis PathwayImmunogold LabelingNeuronal ApoptosisMitochondrial PermeabilityRat Model
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