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Overview of Immunoelectron Microscopy
Applied Microscopy 2018;48:87-95
Published online December 28, 2018
© 2018 Korean Society of Microscopy.

Chang-Hyun Park*, Hong Lim Kim1, Byung-Joon Chang2, Sang Hoon Lee3, Byung Soo Chang4, Chun-Sik Bae5, Ik-Hyun Cho6, Dong Heui Kim7, Jung-Mi Han2, Ji Eun Na8, Byung-Jin Choi, Sang-Sik Kim, Hyun-Wook Kim, Jee-Woong Kim9, Im Joo Rhyu8, and Chang-Sub Uhm8

Medical Science Research Center, Korea University College of Medicine, Seoul 02841, Korea, 1Integrative Research Support Center, The Catholic University of Korea, Seoul 06591, Korea, 2College of Veterinary Medicine, Konkuk University, Seoul 05029, Korea, 3Korea Brain Research Institute, Daegu 41062, Korea, 4Department of Cosmetology, Hanseo University, Seosan 31962, Korea, 5College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Korea, 6Department of Convergence Medical Science, College of Korean Medicine, Kyung Hee University, Seoul 02447, Korea, 7Department of Environmental Medical Biology, Yonsei University Wonju College of Medicine, Wonju 26426, Korea, 8Department of Anatomy, Korea University College of Medicine, Seoul 02841, Korea, 9Division of Biosafety Evaluation and Control, Korea National Institute of Health, Cheongju 28159, Korea
Correspondence to: Park CH, http://orcid.org/0000-0002-7306-035X, Tel: +82-2-2286-1323, Fax: +82-2-2286-1329, E-mail: kmemno1@empal.com
Received July 24, 2018; Revised November 5, 2018; Accepted November 11, 2018.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Immunoelectron microscopy using an antigen-antibody reaction in an electron microscope is a very useful tool to identify the components of a tissue in an electron microscope. Many researchers also use immunoelectron microscopy. Nonetheless, immunoelectron microscopy is rarely introduced systematically, and immunoelectron microscopy can be carried out without fully understanding the principles, and cases of poor understanding can often be seen in the vicinity. Therefore, in order to make it easier to understand, we will first introduce the principles of immunoelectron microscopy and describe practical methods.

Keywords : Pre-embedding, Post-embedding, Immuno cytochemistry, Correlative light and electron microscopy
Figures
Fig. 1. Structure of the immunoglobulin G molecule.
Fig. 2. Comparison of pre-embedding and post-embedding.
Fig. 3. Immunolabelling was performed with a primary antibody: Chemicon MAB922 & secondary antibody: Gold-conjugated (15 nm) protein A (supplied by Prof. Su Jin Kim), and then refinement & osmication and routine processing for scanning electron microscope (SEM), freeze drying and observed by field emission SEM (S-4700; Hitachi). The white spots in the picture are 15 nm gold. In the transmission electron microscope, the gold particles appear black because they interfere with the transmission of electrons ().
Fig. 4. Electron micrographs showing osteopontin-labeled mitochondria (arrows) using immunoperoxidase electron microscopy in the 3-nitropropionic acid-injured lesion core in rat brain. Scale bar=1 μm.
Fig. 5. (A, B) Electron micrograph showing nestin-positive cell associated with capillaries at 3 days post-ischemia by using immunogold/silver electron microscopy. Higher magnification (B) of the boxed area in Fig. 5A shows the silver grains (arrow) were exclusively localized along the bundles of intermediate filaments. Scale bars=2 μm (A), 0.4 μm (B).
Fig. 6. (7) Post-embedding immunoelectron micrograph of neurofascin in 14 days old rat sciatic nerve. Unmyelinated fiber (UM) expresses neurofascin strongly in the axolemma (arrowheads), but myelinated fiber (M) doesn’t express neurofascin in the axolemma at all. SC, Schwann cell cytoplasm. Scale bar=200 nm. (8) Neurofascin immunoreactive gold particles are labeled in the outer mesaxon (arrowheads). My, compact myelin sheath. Scale bar=200 nm. (9) Node (N) and paranodal loops (PL) are shown. Many neurofascin immunoreactive gold particles are localized in the nodal axolemma (arrows). Neurofascin expression was revealed in the paranodal loops as well. Scale bar=500 nm. (10) Post-embedding immunoelectron micrograph of neurofascin in 5 days old rat sciatic nerve. Neurofascin immunoreactive gold particles are labeled in the inner mesaxon (arrowhead). Scale bar=200 nm. (11) Post-embedding immunoelectron micrograph of neurofascin in 14 days old rat sciatic nerve. Neurofascin immunoreactive gold particles are labeled in the Schmidt-Lantermann incisures (arrowheads). Scale bar=200 nm. (12) Non-compact myelin layer which in composed of apposing Schwann cell membranes expresses neurofascin immunoreactivity (arrowheads), but compact myelin layer doesn’t express neurofascin at all. Scale bar=200 nm. Adapted from the article of (Korean J. Electron Microsc. , 131–140) with original copyright holder’s permission.
Fig. 7. (A) Confocal microscopic double-labeled image with osteopontin and mitochondria marker NDUFV2. Higher magnification the boxed area in Fig. 7A show the corresponding transmission electron microscopic image obtained from the same field in the 3-nitropropionic acid (3NP)-injured lesion core (B–D). Scale bars=5 μm (A), 10 μm (B–D).
Tables

Relative affinity of protein A, G, and A/G

Primary antibody Protein A gold Protein C gold Protein A/G gold
Rabbit IgG +++ +++ +++
Mouse IgG ++ ++ ++
IgM +/− +/− +/−
IgA +/− +/− +/−
Rat IgG +/− + +++
Human IgG1 +++ +++ +++
IgG2 +++ +++ +++
IgG3 +/− +++ +++
IgG4 +++ +++ +++
IgM +
IgA +
Guinea pig IgG ++ ++ ++
Goat IgG +/− ++ +++
Bovine IgG ++ ++ ++
Sheep IgG +/− ++ +++
Chicken IgG +/− + +/−

Ig, immunoglobulin.


Selection of gold particles according to observation method

Condition Gold particle size (nm) Remarks
TEM
 Low magnification 15~30
 High magnification 5~10 10 nm particles are most commonly usedSilver enhancement if needed
 Ultra-sensitive labeling 1 Silver enhancement required
 Pre-embedding labeling 1 Silver enhancement required
SEM 20~30 Unenhanced observation
5 Enhanced observation

Characteristics of fixative

Type of fixative Structure preservation Antigenic preservation Application field
Coagulative (cross linking)
 Formaldehyde ++ ++ Tissues
 Glutaraldehyde +++ + Tissues
Additive (precipitating)
 Acetone + + Cells
 Methanol + + Cells
Mixed
 Formal acetone ++ + Cells
 Picric acid ++ + Tissues

References
  1. Chang, BH, You, KH, Lee, JH, Cho, IH, Bae, CS, Park, CH, Han, JM, Choe, NH, and Chang, BJ (2006). A study on the localization of neurofascin in the myelinated rat sciatic nerve fibers. Korean J Electron Microsc. 36, 131-140.
  2. Goldberg, MW (2016). High-resolution scanning electron microscopy and immuno-gold labeling of the nuclear lamina and nuclear pore complex. Methods Mol Biol. 1411, 441-459.
    Pubmed CrossRef
  3. Melo, RC, Morgan, E, Monahan-Earley, R, Dvorak, AM, and Weller, PF (2014). Pre-embedding immunogold labeling to optimize protein localization at subcellular compartiments and membrane microdomains of leukocytes. Nat Protoc. 9, 2382-2394.
    Pubmed KoreaMed CrossRef


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